High-Throughput Screen For Identifying Selective Persistent Sodium Channels Channel Blockers

ABSTRACT

A method for identifying a selective persistent Na +  channel blocker by measuring the ability of the blocker to reduce or inhibit a persistent Na +  current to a greater degree than a transient Na +  current. Aspects of the present method provide Na +  depletion/repletion methods for identifying a selective blocker of a persistent Na +  channel, hyperpolarization methods for identifying a blocker of a persistent Na+ channel, and Na/K ATPase pump inhibitor methods for identifying a selective blocker of a persistent Na +  channel.

This application is a divisional that claims priority pursuant 35 U.S.C.§ 120 to U.S. patent application Ser. No. 11/313,450, filed Dec. 19,2005, continuation-in-part application that claims priority pursuant to35 U.S.C. § 120 to U.S. Pat. No. 6,991,910, filed Nov. 20, 2001, each ofwhich is hereby incorporated by reference in its entirety.

All of the patents and publications cited in this application are herebyincorporated by reference in their entirety.

The lipid bilayer membrane of all cells forms a barrier that is largelyimpermeable to the flux of ions and water. Residing within the membraneis a superfamily of proteins called ion channels, which provideselective pathways for ion flux. Precisely regulated conductancesproduced by ion channels are required for intercellular signaling andneuronal excitability. In particular, a group of ion channels that openupon depolarization of excitable cells are classified as voltage-gatedand are responsible for electrical activity in nerve, muscle and cardiactissue. In neurons, ion currents flowing through voltage-gated sodiumion (Na⁺) channels are responsible for rapid spike-like actionpotentials. During action potentials the majority of Na⁺ channels openvery briefly. These brief openings result in transient Na⁺ currents.However, a subset of voltage-gated Na⁺ channels does not close rapidly,but remain open for relatively long intervals. These channels thereforegenerate sustained or persistent Na⁺ currents. The balance betweentransient and persistent Na⁺ current is crucial for maintaining normalphysiological function and electrical signaling throughout the entirenervous system.

Over the past 50 years, an increasing number of diseases of the nervoussystem and other excitable tissues have been shown to result from thedysregulation of ion channels. This class of disease has been termedchannelopathies. Aberrant persistent sodium current can contribute tothe development or progression of many channelopathic conditions becausenormal function is disrupted when neurons discharge signalsinappropriately. For example, abnormal persistent sodium current isthought to induce deleterious phenomena, including, e.g., neuropathies,neurodegenerative diseases, movement disorders, cardiac arrhythmia,epileptic seizure, neuronal cell death, behavioral disorders anddementia, see, e.g., Robert S. Kass, The Channelopathies: Novel Insightsinto Molecular and Genetic Mechanisms of Human Disease, 115(8) J. Clin.Invest. 1986-1989 (2005); Alfred L. George, Inherited Disorders ofVoltage-gated Sodium Channels, 115(8) J. Clin. Invest. 1990-1999 (2005);Karin Jurkat-Rott and Frank Lehmann-Horn, Muscle Channelopathies andCritical Points in Functional and Genetic Studies, 115(8) J. Clin.Invest. 2000-2009 (2005); Miriam H. Meisler and Jennifer A. Kearney,Sodium Channel Mutations in Epilepsy and Other Neurological Disorders,115(8) J. Clin. Invest. 2010-2017 (2005); Arthur J. Moss and Robert S.Kass, Long QT Syndrome: from Channels to Cardiac Arrhythmias, 115(8) J.Clin. Invest. 2018-2024 (2005); Christoph Lossin et al., Molecular Basisof an Inherited Epilepsy 34(6) NEURON 877-84 (2002); Peter K. Stys etal., Ionic Mechanisms of Anoxic Injury in Mammalian CNS White Matter:Role of Na ⁺ Channels and Na ⁽⁺⁾—Ca2⁺ Exchanger, 12(2) J. NEUROSCI.430-439 (1992); Peter K. Stys et al., Noninactivating,Tetrodotoxin-Sensitive Na⁺ Conductance in Rat Optic Nerve Axons, 90(15)PROC. NATL. ACAD. SCI. USA, 6976-6980 (1993); and Giti Garthwaite etal., Mechanisms of Ischaemic Damage to Central White Matter Axons: AQuantitative Histological Analysis Using Rat Optic Nerve, 94(4)NEUROSCIENCE 1219-1230 (1999). For example, in the case of theneuropathies embraced by epilepsy, there can be a brief electrical“storm” arising from neurons that are inherently unstable because of agenetic defect as in various types of inherited epilepsy, or fromneurons made unstable by metabolic abnormalities such as low bloodglucose, or alcohol. In other cases, the abnormal discharge can comefrom a localized area of the brain, such as in patients with epilepsycaused by head injury or brain tumor. In the case of ischemic injuries,such as, e.g., cerebral ischemia and myocardial ischemia, there can beprolonged electrical activity arising from neurons in which persistentsodium channel expression or activity is increased. Such aberrantelectrical activity can cause or contribute to neuronal death, which canlead to debilitating injury or death of an individual. Aberrantelectrical activity also can contribute to neurodegenerative disorderssuch as, without limitation, Parkinson's disease, Alzheimer's disease,Huntington's disease, amyotrophic lateral sclerosis and multiplesclerosis. Thus, aberrant persistent sodium current can contribute todevelopment or progression of pathological conditions by collapsing thenormal cell transmembrane gradient for sodium, leading to reverseoperation of the sodium-calcium exchanger, and resulting in an influx ofintracellular calcium, which injures the axon, see, e.g., Stys et al.,supra, (1992). Therefore, selective reduction in the expression oractivity of sodium channels capable of mediating persistent currentrelative to any reduction in normal voltage-gated (transient) sodiumcurrent can be useful for treating channelopathic conditions associatedwith increased persistent sodium current.

Recent evidence has revealed that increased activity from persistent Na⁺channels may be responsible for the underlying basis of chronic pain,see e.g., Fernando Cervero & Jennifer M. A. Laird, Role of Ion Channelsin Mechanisms Controlling Gastrointestinal Pain Pathways, 3(6) CURR.OPIN. PHARMACOL. 608-612 (2003); Joel A. Black et al., Changes in theExpression of Tetrodotoxin-Sensitive Sodium Channels Within Dorsal RootGanglia Neurons in Inflammatory Pain, 108(3) PAIN 237-247 (2004) and LiYunru et al., Role of Persistent Sodium and Calcium Currents inMotoneuron Firing and Spasticity in Chronic Spinal Rats, 91(2) J.NEUROPHYSIOL. 767-783 (2004). Alterations in persistent sodium channelexpression and/or function has a profound effect on the firing patternof neurons in both the peripheral and central nervous systems. Forexample, injury to sensory primary afferent neurons often results inrapid redistribution of persistent sodium channels along the axon anddendrites and in abnormal, repetitive discharges or exaggeratedresponses to subsequent sensory stimuli. Such an exaggerated response isconsidered to be crucial for the incidence of spontaneous pain in theabsence of external stimuli that is characteristic of chronic pain. Inaddition, inflammatory pain is associated with lowered thresholds ofactivation of nociceptive neurons in the periphery and alteredpersistent sodium channel function is thought to underlie aspects ofthis phenomenon. Likewise, neuropathic pain states resulting fromperipheral nerve damage is associated with altered persistent sodiumchannel activity and ectopic action potential propagation. Therefore,selective reduction in the expression or activity of sodium channelscapable of mediating persistent current relative to any reduction innormal voltage-gated (transient) sodium current can be useful fortreating chronic pain conditions associated with increased persistentsodium current.

Besides their importance under physiological conditions, Na⁺ channelsare also important under pathophysiological situations. For example theyappear play a role in epileptic seizures, cardiac arrhythmias, andischemia/hypoxia-induced cardiac and neuronal cell death (Taylor et al,1997; Ragsdale et al, 1998). Importantly, the persistent Na⁺ currentappears to play a major role in generating the above mentioned cellularabnormalities (Stys, 1998; Taylor et al, 1997). For example persistentNa⁺ current is unregulated in both cardiac and neuronal cells duringhypoxia (Saint et al, 1996; Hammarstrom, 1998) and may ultimately leadto overload of cell Na⁺ and calcium, conditions leading to cell death(Stys, 1998). Blockers of voltage-gated Na⁺ channels have been shown tobe effective in ameliorating cellular dysfunctions and death resultingfrom errant operation of voltage-gated sodium channels (Stys, 1998).However, in many cases these blockers inhibit both the normalinactivating (transient) and non-inactivating (persistent) Na⁺ channelsto the same extent. Significant block of normal transient Na⁺ channelscould seriously compromise cellular and organ function or may even causedeath. Thus assuming that the persistent Na⁺ current is the therapeutictarget, it is important to develop drugs that will block this componentof Na⁺ current but not the normal transient. However, in order todiscern whether a compound selectively blocks the persistent over thetransient Na⁺ current conventional electrophysiological methods such aswhole cell patch clamping or voltage clamping in oocyte preparationsmust be performed (Marty and Neher, 1995; Shih et al, 1998).

Thus, there exists a need for new screening methods that can be used toidentify persistent Na⁺ channel blockers useful for treatingchannelopathies and chronic pain. The present invention satisfies thisneed and provides related advantages as well, such as, e.g.,high-throughput screens for identifying voltage-gated Na⁺ channelblockers that selectively reduce or prevent persistent Na⁺ currents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a schematic of a basic ion channel mechanism. The topillustration shows a K⁺ channel and K⁺ ion flow, a transient Na⁺ channeland ion flow, a persistent Na⁺ channel and ion flow and a Na/K ATPasepump and K⁺ and Na⁺ ion flow. While drawn as separate channels here, thesame Na⁺ channel can have both transient and persistent currentproperties. The bottom panels show a current recording from a cellcontaining a transient Na⁺ current (left panel) and a recording from acell containing both transient and persistent Na⁺ currents (rightpanel). Current vs. time is plotted for a voltage-gated Na⁺ current. Inthe left panel, the initial transient current is shown during a 2 msecdepolarization. In the right panel, the initial transient current and asmaller sustained persistent current are shown during a 200 msecdepolarization.

FIG. 2 shows a schematic of a Na⁺ depletion/repletion protocol. Foursteps integral to the assay are illustrated. 1) Providing Sample: A cellcontaining the principal components of the assay—a K⁺ channel andchannel(s) capable of producing transient and persistent Na⁺ currentsare incubated in Na⁺—free solution containing a voltage-sensitive dye(Dye) and a test compound (Blocker). 2) Depolarizing the cell: A smallaliquot of solution containing concentrated K⁺ is added to the solutionto initiate a depolarization of the membrane sufficient to activate theNa⁺ channels. In the absence of external Na⁺ to act as charge carrierthrough the Na⁺ channels only small background K⁺-induced depolarizationand fluorescence change is produced (see also FIG. 3). 3) Generatingcurrent and detecting fluorescence emitted in the absence of aneffective persistent Na⁺ channel blocker: Following and intervalsufficient to allow the closure of transient Na⁺ channels, an aliquot ofsolution containing concentrated Na⁺ sufficient to raise the externalNa⁺ concentration to physiological levels is added. In the absence of aneffective persistent Na⁺ channel blocker, Na⁺ ions acting as a chargecarrier through persistent Na⁺ channels produce a depolarization of thecell membrane and a subsequent change in fluorescence of thevoltage-sensitive dye. 4) Generating current and detecting fluorescenceemitted in the presence of an effective persistent Na⁺ channel blocker:With solution additions as in (3) above, except that the solution nowcontains an effective blocker of persistent Na⁺ channels, Na⁺ ions areprevented from entering the cell, no depolarization occurs and no changein fluorescence is observed.

FIG. 3 shows a graphic depiction of an emitted fluorescence readoutusing a Na⁺ depletion/repletion protocol. Fluorescence in relativefluorescence units vs. time is plotted for a voltage-gated Na⁺ current.The recording of a molecule exhibiting a blocking activity of apersistent Na⁺ current is indicated by a black line. A control samplewhich lacks blocking activity of a persistent Na⁺ current is indicatedby a dashed line.

FIG. 4 shows a schematic of a hyperpolarization protocol. Thehyperpolarization protocol illustrated here is similar to thedepletion/repletion protocol shown in FIG. 2, except that test cell isengineered to have approximately equal K⁺ and Na⁺ conductances and aresting potential midway between the equilibrium potentials for K⁺ andNa⁺. This resting potential would also be engineered to result in theinactivation of transient Na⁺ channels. Three steps integral to theassay are illustrated. 1) Providing Sample: A cell containing theprincipal components of the assay—a K⁺ channel and channel capable ofproducing persistent Na⁺ currents are incubated in Na⁺-containingsolution which also contains a voltage-sensitive dye (Dye). In this caseNa⁺ ions acting as charge carriers through the open persistent Na⁺channels will result in a steady-state depolarization of the cellmembrane and significant fluorescent emission from the voltage-sensitivedye. 2) Adding a potential blocker (ineffective compound) and detectingfluorescence emitted: If the compound is ineffective in blocking thepersistent Na⁺ current, no change in Na⁺ influx, depolarization oremitted fluorescence will occur. 3) Adding a potential blocker(effective compound) and detecting fluorescence emitted: If the compoundis effective in blocking the persistent Na⁺ current, a decrease in Na⁺influx, depolarization and emitted fluorescence will occur.

FIG. 5 shows a graphic depiction of an emitted fluorescence readoutusing a hyperpolarization protocol. Fluorescence in relativefluorescence units vs. time is plotted for a voltage-gated Na⁺ current.The recording of a molecule exhibiting a blocking activity of apersistent Na⁺ current is indicated by a black line. A control samplewhich lacks blocking activity of a persistent Na⁺ current is indicatedby a dashed line.

FIG. 6 shows a schematic of a Na/K pump inhibitor protocol. Two stepsintegral to the assay are illustrated. 1) Adding Ouabain: In a Cl-freephysiological solution ouabain (100 uM) is added to engineered cellswhere G_(K) is at least 20fold>than G_(Na+) persistent. 2) Detectingfluorescence emitted: Inhibition of the Na/K pump causes the cells toexchange extracellular Na⁺ (via influx through persistent Na+ channels)for intracellular K. The loss of K changes E_(K) favoring celldepolarization, reflected as an increase in fluorescence intensity forthe case of an anionic dye. In the presence of a persistent Na+ channelblocker the K-dependent depolarization will be inhibited.

FIG. 7 shows a graphic depiction of an emitted fluorescence readoutusing a Na/K pump inhibitor protocol. Fluorescence in relativefluorescence units vs. time is plotted for a voltage-gated Na⁺ current.The recording of a molecule exhibiting a blocking activity of apersistent Na⁺ current is indicated by a black line. A control samplewhich lacks blocking activity of a persistent Na⁺ current is indicatedby a dashed line.

FIG. 8 shows a schematic of a transient blocker protocol. 1) Fieldstimulation of cells in wells: The engineered cells are placed in wellscontaining an appropriate physiological solution and a pair ofstimulating electrodes capable of passing sufficient current to reachthreshold for action potential initiation. 2) Detection of emittedfluorescence following stimulation to threshold: Depolarization of thecell following the upstroke to the action potential is detected by anincrease in emitted fluorescence. 3) Adding a potential blocker anddetecting emitted fluorescence. If a compound is a persistent Na⁺channel blocker an increase in emitted fluorescence will not bedetected.

FIG. 9 shows a graphic depiction of an emitted fluorescence readoutusing a transient blocker protocol. Fluorescence in relativefluorescence units vs. time is plotted for a voltage-gated Na⁺ current.The recording of a molecule exhibiting a blocking activity of atransient Na⁺ current is indicated by a black line. A control samplewhich lacks blocking activity of a transient Na⁺ current is indicated bya dashed line. A selective persistent Na⁺ channel blocker would lacksignificant blocking activity of a transient Na⁺ current and thus behavemore like a control sample.

FIG. 10 shows inhibition of persistent current-dependent depolarizationby Na⁺ channel blockers. In this assay, cells are resting in wellscontaining a 80 μL solution of 140 mM TEA-MeSO₃ (Na⁺-free box) to whichis added 240 μL solution of 140 mM NaMeSO₃ and 13 mM KMeSO₃ for a finalK⁺ concentration of 10 mM and a final Na⁺ concentration of 110 mM(Na⁺/K⁺-addition). This elicits a robust depolarizing response.Following the resolution of the sodium-dependent depolarization, asecond aliquot of KMeSO₃ is added to the well bringing the final K⁺concentration to 80 mM (high potassium-addition). This addition resultsin a second depolarizing response. Compounds that reduce thesodium-dependent, but not the potassium-dependent depolarizations areselected as persistent sodium channel blockers. Circles indicate thecontrol response with 0.1% DMSO added, triangles show the effects of thesodium channel inhibitor tetracaine (10 μM) and the diamonds show theresponse during the application of a non-specific channel blocker.

FIG. 11 shows data from assays in which the screening window for thepersistent current assay is determined. To evaluate the size of the“screening window,” data was examined from assays in which responses tosodium-dependent depolarization were measured in the presence of 10 μMTetracaine to completely block the sodium-dependent depolarization or inthe presence of a 0.1% DMSO control to obtain a maximum depolarization.Data were binned into histograms and a screening window (Z) wascalculated from the mean and standard deviation for the maximal andminimum values according to the equation:Z=1-(3×STD_(Max)+3×STD_(Min))/(Mean_(Max)−Mean_(Min)). Histograms A, Band C represent data obtained from three different assay plates. Thescreening window for a run was considered adequate 1.0≧Z≧0.5.

FIG. 12 shows sodium current traces before and after the addition of 3μM Compound 1 or 500 nM TTX. HEK cells expressing Na_(v)1.3 channelswere patch clamped in the perforated-patch mode. Currents were elicitedby 200 msec test pulses to 0 mV from a holding potential of −90 mV.

FIG. 13 shows a dose-response curve for Compound 1. The peak amplitudesof transient Na⁺ current (I_(t)) and the steady state amplitude of thepersistent current (I_(p)) were measured at various Compound 1concentrations, normalized to the amplitude of the control currents. Thepercent block was then plotted against drug concentration. Solid linesrepresent fits to the data with the Hill equation. The calculated IC₅₀values and Hill coefficients are as follows: Hill slope, I_(t) is 0.354and I_(p) is 0.733; IC₅₀, I_(t) is 0.167 M and I_(p) is 3.71×10⁻⁶ M.

FIG. 14 shows the use of a single-wavelength dye to measure persistentNa⁺ channel activity to measure fluorescence on the FLIPR-Tetra. (A) Rawfluorescence signals are shown during application of the Na⁺depletion/repletion protocol. Base line measurements in the presence ofa Na⁺-free buffer are shown for the first 4 seconds of the record. Afterestablishing of the base line fluorescence, a depolarizing buffercontaining Na⁺ is applied to the well resulting a an initial rapidincrease in fluorescence followed by a longer sustained increase(Control). In the presence of a saturating concentration of TTX (1 μMTTX) to block all the Na⁺ channel mediated signal, the initial responseis lost and only the sustained non-channel mediated response remains.(B) Subtraction of the TTX resistant response from the control responsereveals the persistent Na⁺ current mediated signal.

FIG. 15 shows typical compound plate layouts for the persistent currentassay on the FLIPR-tetra. (A) In the screening window format a 96-wellcompound plate is organized such that columns 1-5 & 8-12 contain thenormal Na⁺ repletion solution while columns 6-7 contain the Na⁺repletion solution plus 1 μM TTX. (B) For the drug screeningdose-response protocol columns 1-2 & 11-12 were used for positive andnegative controls. The remaining columns contained differentconcentrations of the test compound with the highest concentration incolumn 10 and serial three-fold dilutions from column 9 through 3.

FIG. 16 shows the results of the screening window experiment. (A) Ascreenshot from the FLIPR-Tetra. Plate was loaded as described in FIG.15 A. Data are presented following the subtraction of the non-specific(TTX-resistant) response. (B) Measurement of the peak response in thepresence and absence of 1 μM TTX. Data are displayed as mean ±SD. Z′factor of 0.70 was calculated as described in example 2 demonstrates anacceptable screening window for this assay.

FIG. 17 shows a dose response analysis using the FLIPR-tetra basedpersistent current assay. (A) Illustrates the persistent Na⁺ currentmediated signal in wells loaded as described in FIG. 15 B. Columns 1 and11 illustrate a negative control with no persistent current blocker andcolumns 2 and 12 show positive controls in the presence of 1 μM TTX. Theremaining columns show a dose-response for TTX with increasingconcentrations left to right. (B) The averaged responses from eachcolumn are plotted vs. the time for the TTX dose response. (C) Averageddata for TTX, Lidocaine, and Tetracaine are plotted as a semi-log doseresponse as mean ±SD, the data is fitted by logistic function (lines)and the estimated IC₅₀ values are shown (mean ±SD).

FIG. 18 shows transient Na⁺ currents taken from 4 wells on an IonWorksautomated patch clamp device. Control (Pre-addition) and TTX blocked(100 nM TTX) traces are shown for each cell.

FIG. 19 shows a dose response analysis of the IonWorks transient currentassay. Averaged currents are plotted as a semi-log dose response as mean±SD in the presence of increasing concentrations of TTX. The data isfitted by logistic function (line) and the estimated IC₅₀ value isshown.

DETAILED DESCRIPTION

In the normal functioning of the nervous system, neurons are capable ofreceiving a stimulus, and in response, propagating an electrical signalaway from their neuron cell bodies (soma) along processes (axons). Fromthe axon, the signal is delivered to the synaptic terminal, where it istransferred to an adjacent neuron or other cell. It is the actionpotential that is responsible for electrical transmission in the nervoussystem, and contractility in the heart and skeletal muscle, see, e.g.,Bertil Hille, Ion Channels of Excitable Membranes 3rd ed. SinauerAssociates, Inc. (Sunderland, Mass.) 2001. Generally, under restingconditions, sodium channels are closed until a threshold stimulusdepolarizes the cell membrane. During membrane depolarization, sodiumchannels activate by opening the channel pore briefly (one millisecond)to rapidly generate the upstroke of the action potential and theninactivate by closing the channel pore until the excitable cell returnsto its resting potential and the sodium channels re-enter the restingstate.

Without wishing to be bound by the following, this behavior ofvoltage-gated sodium channels can be understood as follows. Sodiumchannels can reside in three major conformations or states. The restingor closed state predominates at membrane potentials more negative thanapproximately −60 mV. Upon depolarization, the channels open rapidly toallow current flow and, thereby, enter the active state. The transitionfrom resting to active states occurs within a millisecond afterdepolarization to positive membrane potentials. Finally during sustaineddepolarizations of greater than 1-2 ms, the channels enter a secondclosed or inactivated state. Subsequent re-openings of the channelsrequire a recycling of the channels from the inactive to the restingstate, which occurs when the membrane potential returns to negativevalues. This means that membrane depolarization not only opens sodiumchannels but also causes them to close even during sustaineddepolarizations (Hodgkin and Huxley, 1952). Thus normal Na⁺ channelsopen briefly during depolarization and are closed at rest.

However, some Na⁺ channels may be open under resting conditions atrelatively negative membrane potentials and even during sustaineddepolarization (Stys, 1998; Taylor, 1993). These non-inactivating Na⁺channels generate what is known as a persistent Na⁺ current, see FIG. 1.Persistent Na⁺ channels have these properties because they activate(open) at more negative membrane potentials than normal Na⁺ channels andinactivate at more positive potentials (Alonso et al, 1999). This meansthat these persistent Na⁺ channels may be open at membrane potentials asnegative as −80 mV (Stys, 1998) and stay open at potentials as positiveas 0 mV (Alonso, et al, 1999). These persistent Na⁺ channels are thoughtto be involved in synaptic amplification and modification of spikingbehavior and also in the generation of conditions leading to cellulardysfunction (Ragsdale et al, 1998; and Taylor, 1993). This uniqueproperty of persistent Na⁺ channels is exploited in the assays inaccordance with the present invention.

Aspects of the present invention provide Na⁺ depletion/repletion methodsfor identifying a selective blocker of a persistent Na⁺ channel, suchmethod comprising the steps of a) providing a test sample 1 comprising aNa⁺-free physiological solution, a voltage-sensitive fluorescence dye, acell having a K⁺ channel, a transient Na⁺ channel and a persistent Na⁺channel; and a potential Na⁺ channel blocker; b) depolarizing themembrane of the cell in the test sample 1; c) generating a currentthrough the persistent Na⁺ channel by adding Na⁺ to test sample 1 atleast 10 msec after step (b); d) detecting fluorescence emitted by thevoltage-sensitive dye in test sample 1; e) providing a control sample 1comprising a Na⁺-free physiological solution, a voltage-sensitivefluorescence dye, and a cell having a K⁺ channel, a transient Na⁺channel and a persistent Na⁺ channel; f) depolarizing the membrane ofthe cell in the control sample 1; g) generating a current through thepersistent Na⁺ channel by adding Na⁺ ions to the control sample 1 atleast 10 msec after step (f); h) detecting fluorescence emitted by thevoltage-sensitive dye in the control sample 1; i) comparing the emittedfluorescence from step (d) to the emitted fluorescence from step (h).

Other aspects of the present invention Na⁺ depletion/repletion provide amethod for identifying a selective blocker of a persistent Na⁺ channel,such method comprising the steps of a) providing a test sample 1comprising a Na⁺-free physiological solution, a voltage-sensitivefluorescence dye, a cell having a K⁺ channel, a transient Na⁺ channeland a persistent Na⁺ channel, and a potential Na⁺ channel blocker; b)depolarizing the membrane of the cell in the test sample 1; c)generating a current through the persistent Na+ channel by adding Na⁺ totest sample 1 at least 10 msec after step (b); d) detecting fluorescenceemitted by the voltage-sensitive dye in test sample 1; e) providing acontrol sample 1 comprising a Na⁺-free physiological solution, avoltage-sensitive fluorescence dye, and a cell having a K⁺ channel, atransient Na⁺ channel and a persistent Na⁺ channel; f) depolarizing themembrane of the cell in the control sample 1; g) generating a currentthrough the persistent Na⁺ channel by adding Na⁺ ions to the controlsample 1 at least 10 msec after step (f); h) detecting fluorescenceemitted by the voltage-sensitive dye in the control sample 1; i)determining the relative emitted fluorescence 1 by comparing the emittedfluorescence from step (d) to the emitted fluorescence from step (h); j)providing a test sample 2 comprising a physiological solution, avoltage-sensitive fluorescence dye, a cell having a K⁺ channel and atransient Na⁺ channel, and a potential Na⁺ channel blocker; k)depolarizing membrane of the cell in test sample 2; l) detecting thefluorescence emitted by the voltage-sensitive dye in test sample 2; m)providing a control sample 2 comprising a physiological solution, avoltage-sensitive fluorescence dye, and a cell having a K⁺ channel and atransient Na⁺ channel; n) depolarizing membrane of the cell in controlsample 2; o) detecting the fluorescence emitted by the voltage-sensitivedye in control sample 2; p) determining a relative emitted fluorescence2 by comparing the emitted fluorescence from step (l) to the emittedfluorescence from step (o); and q) comparing the relative emittedfluorescence 1 in step (i) with the relative emitted fluorescence 2 instep (p).

Other aspects of the present invention provide a hyperpolarizationmethod for identifying a blocker of a persistent Na+ channel, suchmethod comprising the steps of a) providing a test sample 1 comprising aphysiological solution, a voltage-sensitive fluorescence dye, and a cellhaving a K+ channel and a persistent Na+ channel wherein a restingmembrane potential of the cell is approximately halfway between anequilibrium potential of Na+ and an equilibrium potential of K+; b)detecting fluorescence emitted by the voltage-sensitive dye in testsample 1; c) adding a potential Na+ channel blocker to test sample 1; d)detecting fluorescence emitted by the voltage-sensitive dye in the testsample 1; e) comparing the emitted fluorescence from step (b) with theemitted fluorescence from step (d).

Other aspects of the present invention provide a hyperpolarizationmethod for identifying a blocker of a persistent Na+ channel, suchmethod comprising the steps of a) providing a test sample 1 comprising aphysiological solution, a voltage-sensitive fluorescence dye, and a cellhaving a K⁺ channel and a persistent Na⁺ channel wherein a restingmembrane potential of the cell is approximately halfway between anequilibrium potential of Na⁺ and an equilibrium potential of K⁺; b)detecting fluorescence emitted by the voltage-sensitive dye in testsample 1; c) adding a potential Na⁺ channel blocker to test sample 1; d)detecting fluorescence emitted by the voltage-sensitive dye in the testsample 1; e) determining a relative emitted fluorescence 1 by comparingthe emitted fluorescence from step (b) with the emitted fluorescencefrom step (d); f) providing a test sample 2 comprising a physiologicalsolution, a voltage-sensitive fluorescence dye, a cell having a K⁺channel and a transient Na⁺ channel, and a potential Na⁺ channelblocker; g) depolarizing the membrane of the cell in test sample 2; h)detecting the fluorescence emitted by the voltage-sensitive dye in testsample 2; i) providing a control sample 2 comprising a physiologicalsolution, a voltage-sensitive fluorescence dye, and a cell having a K⁺channel and a transient Na⁺ channel; j) depolarizing the membrane of thecell in control sample 2; k) detecting the fluorescence emitted by thevoltage-sensitive dye in control sample 2; l) determining a relativeemitted fluorescence 2 by comparing the emitted fluorescence from step(h) relative to an emitted fluorescence from step (k); and m) comparingthe relative emitted fluorescence in step (e) with the relative emittedfluorescence in step (l).

Other aspects of the present invention provide a Na/K ATPase pumpinhibitor method for identifying a blocker of a persistent Na⁺ channel,such method comprising the steps of a) providing a test sample 1comprising a Cl⁻-free physiological solution, a voltage-sensitivefluorescence dye, a cell having a K⁺ channel and a persistent Na⁺channel wherein a K⁺ conductance of the K⁺ channel is at least 20-foldhigher than a Na⁺ conductance from the persistent Na⁺ channel, and apotential Na⁺ channel blocker; b) depolarizing the membrane of the cellwith a Na/K pump inhibitor to the test sample 1; c) detectingfluorescence emitted by the voltage-sensitive dye in test sample 1; d)providing a control sample 1 comprising a Cl⁻-free physiologicalsolution, a voltage-sensitive fluorescence dye, and a cell having a K⁺channel and a persistent Na⁺ channel wherein a K⁺ conductance of the K⁺channel is at least 20-fold higher than a Na⁺ conductance from thepersistent Na⁺ channel; e) depolarizing the membrane of the cell with aNa/K pump inhibitor to the control sample 1; f) detecting fluorescenceemitted by the voltage-sensitive dye in the control sample 1; g)comparing the emitted fluorescence from step (c) to the emittedfluorescence from step (f).

Other aspects of the present invention provide a Na/K ATPase pumpinhibitor method for identifying a selective blocker of a persistent Na⁺channel, such method comprising the steps of a) providing a test sample1 comprising a Cl⁻-free physiological solution, a voltage-sensitivefluorescence dye, a cell having a K⁺ channel and a persistent Na⁺channel wherein a K⁺ conductance of the K⁺ channel is at least 20-foldhigher than a Na⁺ conductance from the persistent Na⁺ channel, and apotential Na⁺ channel blocker; b) depolarizing the membrane of the cellwith a Na/K pump blocker to the test sample 1; c) detecting fluorescenceemitted by the voltage-sensitive dye in test sample 1; d) providing acontrol sample 1 comprising a Cl⁻-free physiological solution, avoltage-sensitive fluorescence dye, and a cell having a K⁺ channel and apersistent Na⁺ channel wherein a K⁺ conductance of the K⁺ channel is atleast 20-fold higher than a Na⁺ conductance from the persistent Na⁺channel; e) depolarizing the membrane of the cell with a Na/K pumpblocker to the control sample 1; f) detecting fluorescence emitted bythe voltage-sensitive dye in the control sample 1; g) comparing theemitted fluorescence from step (c) to the emitted fluorescence from step(f); h) providing a test sample 2 comprising a physiological solution, avoltage-sensitive fluorescence dye, a cell having a K⁺ channel and atransient Na⁺ channel, and a potential Na⁺ channel blocker; i)depolarizing the membrane of the cell in test sample 2; j) detecting thefluorescence emitted by the voltage-sensitive dye in test sample 2; k)providing a control sample 2 comprising a physiological solution, avoltage-sensitive fluorescence dye, and a cell having a K⁺ channel and atransient Na⁺ channel; l) depolarizing the membrane of the cell incontrol sample 2; m) detecting the fluorescence emitted by thevoltage-sensitive dye in control sample 2; n) comparing the emittedfluorescence from step (j) relative to an emitted fluorescence from step(m); and o) comparing the difference in step (g) with the difference instep (n).

Aspects of the present invention provide, in part, a selectivepersistent Na⁺ current blocker. As used herein, the term “persistent Na⁺current blocker” means any molecule that for at least one particulardose can reduce or prevent a persistent Na⁺ current. As used herein, theterm “selective persistent Na⁺ current blocker” means any molecule thatfor at least one particular dose can selectively reduce or prevent apersistent Na⁺ current as compared to a transient Na⁺ current. As usedherein, the term “selective” means to have a unique effect or influenceor reacting in only one way or with only one thing. It is envisionedthat a selective persistent Na⁺ channel blocker can modulate apersistent Na⁺ current derived from at least one persistent Na⁺ channelin an antagonistic manner by reducing or preventing a persistent Na⁺current. It is further envisioned that a selective persistent Na⁺channel blocker acting in an antagonistic manner can do so in acompetitive or non-competitive way. Non-limiting examples of a selectivepersistent Na⁺ channel blocker acting in an antagonistic manner include,e.g., a persistent Na⁺ channel pan-antagonist that reduces or preventspersistent Na⁺ current generated from all persistent Na⁺ channelsubunits, a persistent Na⁺ channel-selective antagonist that reduces orprevents persistent Na⁺ current generated from a subgroup of persistentNa⁺ channel subunits, and a persistent Na⁺ channel-specific antagonistthat reduces or prevents persistent Na⁺ current generated from only onepersistent Na⁺ channel subunit.

In an aspect of this embodiment, a selective persistent Na⁺ currentblocker prevents persistent Na⁺ current but does not affect a transientNa⁺ current. In aspects of this embodiment, a selective persistent Na⁺current blocker prevents persistent Na⁺ current and affects, e.g., atmost 5% of a transient Na⁺ current, at most 10% of a transient Na⁺current, at most 15% of a transient Na⁺ current, at most 20% of atransient Na⁺ current or at most 25% of a transient Na⁺ current. Inother aspects of this embodiment, a selective persistent Na⁺ currentblocker reduces a persistent Na⁺ current by, e.g., at least 50%, atleast 60%, at least 70%, at least 80%, at least 90% or at least 95%, andaffects, e.g., at most 5% of a transient Na⁺ current, at most 10% of atransient Na⁺ current, at most 15% of a transient Na⁺ current, at most20% of a transient Na⁺ current or at most 25% of a transient Na⁺current.

In aspects of this embodiment, a selective persistent Na⁺ currentblocker can reduce a persistent Na⁺ current by, e.g., at least 20-foldmore than transient sodium current is reduced, at least 30-fold morethan transient sodium current is reduced, at least 40-fold more thantransient sodium current is reduced, at least 50-fold more thantransient sodium current is reduced, at least 60-fold more thantransient sodium current is reduced, at least 70-fold more thantransient sodium current is reduced, at least 80-fold more thantransient sodium current is reduced, at least 90-fold more thantransient sodium current is reduced or at least 100-fold more thantransient sodium current is reduced. In yet other aspects of thisembodiment, a selective persistent Na⁺ current blocker can reduce apersistent Na⁺ current by, e.g., at least 100-fold more than transientsodium current is reduced, at least 200-fold more than transient sodiumcurrent is reduced, at least 300-fold more than transient sodium currentis reduced, at least 400-fold more than transient sodium current isreduced, at least 500-fold more than transient sodium current isreduced, at least 600-fold more than transient sodium current isreduced, at least 700-fold more than transient sodium current isreduced, at least 800-fold more than transient sodium current isreduced, at least 900-fold more than transient sodium current is reducedor at least 1000-fold more than transient sodium current is reduced.

In an embodiment, selective persistent Na⁺ current blocker can be apersistent Na⁺ channel pan-antagonist. In an aspect of this embodiment,a persistent Na⁺ channel pan-antagonist prevents persistent Na⁺ currentgenerated from all persistent Na⁺ channel subunits but does not affect atransient Na⁺ current. In aspects of this embodiment, a persistent Na⁺channel pan-antagonist prevents persistent Na⁺ current generated fromall persistent Na⁺ channel subunits and affects, e.g., at most 5% of atransient Na⁺ current, at most 10% of a transient Na⁺ current, at most15% of a transient Na⁺ current, at most 20% of a transient Na⁺ currentor at most 25% of a transient Na⁺ current. In other aspects of thisembodiment, a persistent Na⁺ channel pan-antagonist reduces persistentNa⁺ current generated from all persistent Na⁺ channel subunits by, e.g.,at least 50%, at least 60%, at least 70%, at least 80%, at least 90% orat least 95%, and affects, e.g., at most 5% of a transient Na⁺ current,at most 10% of a transient Na⁺ current, at most 15% of a transient Na⁺current, at most 20% of a transient Na⁺ current or at most 25% of atransient Na⁺ current.

In aspects of this embodiment, a persistent Na⁺ channel pan-antagonistcan reduce persistent Na⁺ current generated from all persistent Na⁺channel subunits by, e.g., at least 20-fold more than transient sodiumcurrent is reduced, at least 30-fold more than transient sodium currentis reduced, at least 40-fold more than transient sodium current isreduced, at least 50-fold more than transient sodium current is reduced,at least 60-fold more than transient sodium current is reduced, at least70-fold more than transient sodium current is reduced, at least 80-foldmore than transient sodium current is reduced, at least 90-fold morethan transient sodium current is reduced or at least 100-fold more thantransient sodium current is reduced. In yet other aspects of thisembodiment, a persistent Na⁺ channel pan-antagonist can reduce apersistent Na⁺ current generated from all persistent Na⁺ channelsubunits by, e.g., at least 100-fold more than transient sodium currentis reduced, at least 200-fold more than transient sodium current isreduced, at least 300-fold more than transient sodium current isreduced, at least 400-fold more than transient sodium current isreduced, at least 500-fold more than transient sodium current isreduced, at least 600-fold more than transient sodium current isreduced, at least 700-fold more than transient sodium current isreduced, at least 800-fold more than transient sodium current isreduced, at least 900-fold more than transient sodium current is reducedor at least 1000-fold more than transient sodium current is reduced.

In an embodiment, selective persistent Na⁺ current blocker can be apersistent Na⁺ channel-selective antagonist. In an aspect of thisembodiment, a persistent Na⁺ channel-selective antagonist preventspersistent Na⁺ current generated from a subgroup of persistent Na⁺channel subunits but does not affect a transient Na⁺ current. In aspectsof this embodiment, a persistent Na⁺ channel-selective antagonistprevents persistent Na⁺ current generated from a subgroup of persistentNa⁺ channel subunits and affects, e.g., at most 5% of a transient Na⁺current, at most 10% of a transient Na⁺ current, at most 15% of atransient Na⁺ current, at most 20% of a transient Na⁺ current or at most25% of a transient Na⁺ current. In other aspects of this embodiment, apersistent Na⁺ channel-selective antagonist reduces persistent Na⁺current generated from a subgroup of persistent Na⁺ channel subunits by,e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least90% or at least 95%, and affects, e.g., at most 5% of a transient Na⁺current, at most 10% of a transient Na⁺ current, at most 15% of atransient Na⁺ current, at most 20% of a transient Na⁺ current or at most25% of a transient Na⁺ current.

In aspects of this embodiment, a persistent Na⁺ channel-selectiveantagonist can reduce persistent Na⁺ current generated from a subgroupof persistent Na⁺ channel subunits by, e.g., at least 20-fold more thantransient sodium current is reduced, at least 30-fold more thantransient sodium current is reduced, at least 40-fold more thantransient sodium current is reduced, at least 50-fold more thantransient sodium current is reduced, at least 60-fold more thantransient sodium current is reduced, at least 70-fold more thantransient sodium current is reduced, at least 80-fold more thantransient sodium current is reduced, at least 90-fold more thantransient sodium current is reduced or at least 100-fold more thantransient sodium current is reduced. In yet other aspects of thisembodiment, a persistent Na⁺ channel-selective antagonist can reduce apersistent Na⁺ current generated from a subgroup of persistent Na⁺channel subunits by, e.g., at least 100-fold more than transient sodiumcurrent is reduced, at least 200-fold more than transient sodium currentis reduced, at least 300-fold more than transient sodium current isreduced, at least 400-fold more than transient sodium current isreduced, at least 500-fold more than transient sodium current isreduced, at least 600-fold more than transient sodium current isreduced, at least 700-fold more than transient sodium current isreduced, at least 800-fold more than transient sodium current isreduced, at least 900-fold more than transient sodium current is reducedor at least 1000-fold more than transient sodium current is reduced.

In an embodiment, selective persistent Na⁺ current blocker can be apersistent Na⁺ channel-specific antagonist. In an aspect of thisembodiment, a persistent Na⁺ channel-specific antagonist preventspersistent Na⁺ current generated from only one persistent Na⁺ channelsubunit but does not affect a transient Na⁺ current. In aspects of thisembodiment, a persistent Na⁺ channel-specific antagonist preventspersistent Na⁺ current generated from only one persistent Na⁺ channelsubunits and affects, e.g., at most 5% of a transient Na⁺ current, atmost 10% of a transient Na⁺ current, at most 15% of a transient Na⁺current, at most 20% of a transient Na⁺ current or at most 25% of atransient Na⁺ current. In other aspects of this embodiment, a persistentNa⁺ channel-specific antagonist reduces persistent Na⁺ current generatedfrom only one persistent Na⁺ channel subunits by, e.g., at least 50%, atleast 60%, at least 70%, at least 80%, at least 90% or at least 95%, andaffects, e.g., at most 5% of a transient Na⁺ current, at most 10% of atransient Na⁺ current, at most 15% of a transient Na⁺ current, at most20% of a transient Na⁺ current or at most 25% of a transient Na⁺current.

In aspects of this embodiment, a persistent Na⁺ channel-specificantagonist can reduce persistent Na⁺ current generated from only onepersistent Na⁺ channel subunits by, e.g., at least 20-fold more thantransient sodium current is reduced, at least 30-fold more thantransient sodium current is reduced, at least 40-fold more thantransient sodium current is reduced, at least 50-fold more thantransient sodium current is reduced, at least 60-fold more thantransient sodium current is reduced, at least 70-fold more thantransient sodium current is reduced, at least 80-fold more thantransient sodium current is reduced, at least 90-fold more thantransient sodium current is reduced or at least 100-fold more thantransient sodium current is reduced. In yet other aspects of thisembodiment, a persistent Na⁺ channel-specific antagonist can reduce apersistent Na⁺ current generated from only one persistent Na⁺ channelsubunits by, e.g., at least 100-fold more than transient sodium currentis reduced, at least 200-fold more than transient sodium current isreduced, at least 300-fold more than transient sodium current isreduced, at least 400-fold more than transient sodium current isreduced, at least 500-fold more than transient sodium current isreduced, at least 600-fold more than transient sodium current isreduced, at least 700-fold more than transient sodium current isreduced, at least 800-fold more than transient sodium current isreduced, at least 900-fold more than transient sodium current is reducedor at least 1000-fold more than transient sodium current is reduced.

Aspects of the present invention provide, in part, a test sample and acontrol sample. As used herein, the term “test sample” means a samplecomprising a potential persistent Na⁺ channel blocker. As used herein,the term “potential persistent Na⁺ channel blocker” means any moleculethat is to be tested for its ability to reduce or prevent a persistentNa⁺ current derived from at least one persistent Na⁺ channel. Apotential persistent Na⁺ channel blocker can be an inorganic molecule oran organic molecule. As used herein, the term “control sample” means asample of the same or similar type as the test sample under the sameconditions but which does not contain a potential persistent Na⁺ channelblocker. In addition, a control sample may comprise a defined moleculeknown not to be a persistent Na⁺ channel blocker (a negative controlmolecule) or a defined molecule known to be a persistent Na⁺ channelblocker (a positive control molecule). One skilled in the artunderstands that a variety of control samples are useful in the methodsof the invention and that a control sample can be a positive controlsample or a negative control sample.

Aspects of the present invention provide, in part, a physiologicalsolution. As used herein, the term “physiological solution” means asolutioned solution comprising physiological concentrations of sodium,potassium, magnesium, calcium and chloride. It is also envisioned thatany and all physiological solutions that allow an electrical current tobe measured from the solution can be used in methods disclosed in thepresent specification. Optionally, a physiological solution can containinhibitors for other ion conductances, such as, e.g., a concentration ofcadmium that prevents or reduces calcium current. A physiologicalsolution can be varied as appropriate by one skilled in the art andgenerally depend, in part, on the assay protocol, the cell or thedetection method employed. Ion concentration can be varied asappropriate by one skilled in the art and generally depend, in part, onthe buffering capacity of a particular buffer being used and thedetection means employed.

Aspects of the present invention provide, in part, a Na⁺-freephysiological solution. As used herein, the term “Na⁺-free physiologicalsolution” means a buffered solution comprising physiologicalconcentrations of a non-permeant sodium substitute, potassium,magnesium, calcium and chloride. It is also envisioned that any and allNa⁺-free physiological solutions that allow an electrical current to bemeasured from the solution can be used in methods disclosed in thepresent specification. A non-permeant sodium substitute substitutes Na⁺with an analog cation molecule, such as, e.g., TEA or NMDG⁺. It is alsoenvisioned that any and all Na⁺-free physiological solutions that allowan electrical current to be measured from the solution can be used inmethods disclosed in the present specification. Optionally, a Na⁺-freesolution can contain inhibitors for other ion conductances, such as,e.g., a concentration of cadmium that prevents or reduces calciumcurrent. A Na⁺-free physiological solution can be varied as appropriateby one skilled in the art and generally depend, in part, on the assayprotocol, the cell or the detection method employed. Ion concentrationcan be varied as appropriate by one skilled in the art and generallydepend, in part, on the buffering capacity of a particular buffer beingused and the detection means employed.

Aspects of the present invention provide, in part, a Cl⁻-freephysiological solution. As used herein, the term “Cl⁻-free physiologicalsolution” means a buffered solution comprising physiologicalconcentrations of a non-permeant chloride substitute, potassium,magnesium, calcium and sodium. It is also envisioned that any and allCl⁻-free physiological solutions that allow an electrical current to bemeasured from the solution can be used in methods disclosed in thepresent specification. A non-permeant chloride substitute substitutesCl⁻ with an analog molecule, such as, e.g., gluconate, aspartate,glutamate, cyclamate and methanesulfonate. It is also envisioned thatany and all Cl⁻-free physiological solutions that allow an electricalcurrent to be measured from the solution can be used in methodsdisclosed in the present specification. Optionally, a Cl⁻-free solutioncan contain inhibitors for other ion conductances, such as, e.g., aconcentration of cadmium that prevents or reduces calcium current. ACl⁻-free physiological solution can be varied as appropriate by oneskilled in the art and generally depend, in part, on the assay protocol,the cell or the detection method employed. Ion concentration can bevaried as appropriate by one skilled in the art and generally depend, inpart, on the buffering capacity of a particular buffer being used andthe detection means employed.

A physiological solution, Na⁺-free physiological solution or Cl⁻-freephysiological solution can be buffered, e.g.,2-amino-2-hydroxymethyl-1,3-propanediol (Tris) buffers; Phosphatebuffers, such as, e.g., potassium phosphate buffers and sodium phosphatebuffers; Good buffers, such as, e.g.,piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES),N,N′-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES),3-(N-morpholino) propanesulfonic acid (MOPS),N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),piperazine-N,N′-bis(2-hydroxypropanesulfonic acid) (POPSO),N-tris(hydroxymethyl) methylglycine (Tricine),N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS),3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid(AMPSO), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO),and 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS); saline buffers,such as, e.g., Phosphate-buffered saline (PBS), HEPES-buffered saline,Tris-buffered saline (TBS) and Ringer's. Thus, aspects of thisembodiment may include a buffer concentration of, e.g., at least 1 mM,at least 5 mM, at least 10 mM, at least 20 mM, at least 30 mM, at least40 mM, at least 50 mM, at least 60 mM, at least 70 mM, at least 80 mM,at least 90 mM, or at least 100 mM. Non-limiting examples of how to makeand use specific buffers are described in, e.g., MOLECULAR CLONING, ALABORATORY MANUAL, supra, (2001).

Aspects of the present invention provide, in part, a voltage-sensitivefluorescent dye. The plasma membrane of a cell typically has atransmembrane potential of approximately −70 mV (negative inside) as aconsequence of K⁺, Na⁺ and Cl⁻ concentration gradients maintained byactive transport processes. Voltage-sensitive fluorescent dyes candirectly measure changes in membrane potential resulting from thetranslocation of these ions. It is envisioned that any voltage-sensitiveflorescent dye capable of detecting a change in cell membrane potentialcan be used, including, without limitation, coumarin dyes, such as,e.g.,N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoylphosphatidyl-ethanolamine(CC2-DMPE); anionic and hybrid oxonol dyes, such as, e.g., bis-oxonol,oxonol V (bis-(3-phenyl-5-oxoisoxazol-4-yl)pentamethine oxonol), oxonolVI (bis-(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol),bis-(1,3-diethylthiobarbituric acid)trimethine oxonol (DiSBAC₂(3),bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC₄(3),bis-(1,3-dibutylbarbituric acid)pentamethine oxonol (DiBAC₄(5), RH-155(NK3041), RH-479 (JPW1131), RH-482(JPW1132, NK3630), RH-1691, RH-1692,RH-1838 R-1114(WW781), JPW1177 and JPW1245; hemicyanine dyes, such as,e.g., dibutylamino-naphtalene-butylsulfonato-isoquinolinium (BNBIQ);merocyanine dyes, such as, e.g., merocyanine 540, NK2495(WW375) andJPW1124; cationic or zwitterionic styryl dyes, such as, e.g.,di-4-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate(di-4-ANEPPS),di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate(di-8-ANEPPS), di-12-ANEPPS, di-18:2-ANEPPS, di-2-ANEPEQ(JPW1114),di-12-ANEPEQ, di-8-ANEPPQ, di-12-ANEPPQ, di-1-ANEPIA, D-6923(JPW3028),N-(4-sulfobutyl)-4-(6-(4-(dibutylamino)phenyl)hexatrienyl) pyridinium(RH-237),N-(3-triethylammoniumpropyl)-4-(4-(4-(diethylamino)phenyl)butadienyl)pyridiniumdibromide (RH-414),N-(4-sulfobutyl)-4-(4-(4-(dipentylamino)phenyl)butadienyl) pyridinium(RH-421) and RH-437, RH-461, RH-795, JPW1063 and FM1-43; and cationiccarbocyanines and rhodamines, such as, e.g., 3,3′-diethyloxacarbocyanineiodide (DiOC₂(3)), 3,3′-dihexyloxacarbocyanine iodide (DiOC₆(3)),3,3′-dimethyl-naphthoxacarbocyanine iodide (JC-9; DiNOCl(3)),3,3′-dipentyloxacarbocyanine iodide (DiOC₆(3),3,3′-dipropylthiadicarbocyanine iodide (DiSC₃(5)),1,1′,3,3,3′,3′-hexamethylindodicarbocyanine iodide (DilC₁(5)),rhodamine, rhodamine 123,5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanineiodide (CBIC₂(3), tetramethylrhodamine, ethyl ester, perchlorate (TMRE)and tetramethylrhodamine, methyl ester, perchlorate (TMRM). The class ofdye determines factors such as accumulation in cells, response mechanismand toxicity.

Voltage-sensitive fluorescent dyes can also be divided into two generalcategories of based on whether there is a relatively fast intramolecularredistribution of electrons or a relatively slow transmembrane movementof entire dye (Table 1). Fast-response dyes undergo electricfield-driven changes of intramolecular charge distribution in responseto a change in the surrounding electric field. This change in electronicstructure produce corresponding changes in the spectral properties orintensity of their fluorescence. The optical response of these dyes issufficiently fast to detect transient (millisecond) potential changes inexcitable cells, including single neurons, cardiac cells and intactbrains. However, the magnitude of their potential-dependent fluorescencechange is often small; fast-response probes typically show a 2-10%fluorescence change per 100 mV. Non-limiting examples of Fast-responsedyes include, e.g., di-2-ANEPEQ (JPW1114), di-1-ANEPIA, di-8-ANEPPQ,di-12-ANEPPQ, di-4-ANEPPS, di-8-ANEPPS, di-18:2-ANEPPS, RGA-30, RH-155,RH-795, RH-237, RH-421, RH-414 and WW 781.

Slow-response dyes are lipophilic anions or cations that are exhibitpotential-dependent changes in their transmembrane distribution by anelectrophoretic mechanism. Fluorescence changes associated withtransmembrane redistribution result from sensitivity of the dye tointracellular and extracellular environments. The magnitude of theiroptical responses is much larger than that of fast-response probes(typically a 1% fluorescence change per mV). Slow-response probes, whichinclude cationic carbocyanines and rhodamines and anionic oxonols, aresuitable for detecting changes in average membrane potentials ofnon-excitable cells caused by respiratory activity, ion-channelpermeability, drug binding and other factors. Non-limiting examples ofSlow-response dyes include, e.g., DiSBAC₄(3), DiBAC₄(5), DiBAC₄(3),DiOC₅(3), DiOC₆(3), DiSC₃(5), DiOC₂(3), DiNOC₆(3), DiIC₂(5), merocyanine540, Oxonol V, Oxonol VI, rhodamine 123, TMRM, TMRE and CBIC₂(3).

TABLE 1 Voltage-sensitive fluorescent dyes Dye Response AbsorbanceEmission di-2-ANEPEQ (JPW1114) Fast 517 721 di-1-ANEPIA Fast — —di-8-ANEPPQ Fast 516 (467) 721 (631) di-12-ANEPPQ Fast 519 719di-1-ANEPMI Fast — — di-4-ANEPPS Fast 497 (475) 705 (617) di-8-ANEPPSFast 498 713 di-18:2-ANEPPS Fast 501 705 RGA-30 Fast 629 659 RH-155 Fast650 none RH-795 Fast 530 712 RH-237 Fast 528 (506) 782 (687) RH-421 Fast515 (493) 704 (638) RH-414 Fast 532 716 WW781 Fast 605 639 DiSBAC₄(3)Slow 535 560 DiBAC₄(5) Slow 590 616 DiBAC₄(3) Slow 493 516 DiOC₅(3) Slow484 500 DiOC₆(3) Slow 484 501 DiSC₃(5) Slow 651 675 DiOC₂(3) Slow 482497 DiNOC₁(3) Slow 522 535 DiIC₁(5) Slow 638 658 merocyanine 540 Slow555 578 Oxonol V Slow 610 639 Oxonol VI Slow 599 634 rhodamine 123 Slow507 529 TMRM Slow 549 573 TMRE Slow 549 574 CBIC₂(3) Slow 514 529Spectra values are in methanol with values in parenthesis in a membraneenvironment. Absorbance and emission spectra of styryl dyes are atshorter wavelengths in membrane environments than in reference solventssuch as methanol. The difference is typically 20 nm for absorption and80 nm for emission, but varies considerably from one dye to another.Styryl dyes are generally nonfluorescent in water.

Voltage-sensitive fluorescent dyes have been widely used to monitormembrane potential within neuronal and other cell types, see, e.g.,Amiram Grinvald et al., Optical imaging of neuronal activity, 68(4)Physiol. Rev. 1285-1366 (1988); C. R. Lowe and M J. Goldfinch,Solid-phase optoelectronic biosensors, 137 Methods Enzymol. 338-348(1988); and Haralambos E. Katerinopoulos, The coumarin moiety aschromophore of fluorescent ion indicators in biological systems, 10(30)Curr. Pharm. Des. 3835-3852 (2004). Voltage-sensitive fluorescent dyewith high sensitivity and rapidly response to a change in membranepotential and methods for measuring membrane potential using such dyesare well known to those skilled in the art, and are described in, e.g.,lain D. Johnson, Fluorescent Probes for Living Cells 30(3) HISTOCHEM .J. 123-140 (1998); and IMAGING NEURONS: A LABORATORY MANUAL (RafaelYuste, et al., eds., Cold Spring Harbor Laboratory Press, 2000). Inaddition, the methods disclosed in the present specification can takeadvantage of the high temporal and spatial resolution utilized byfluorescence resonance energy transfer (FRET) in the measurement ofmembrane potential by voltage-sensitive dyes as described, see, e.g.,Jesus E. Gonzalez & Roger Y. Tsien, Improved Indicators of Cell MembranePotential That Use Fluorescence Resonance Energy Transfer 4(4) CHEM.BIOL. 269-277 (1997); Roger Y. Tsien & Jesus E. Gonzalez, VoltageSensing by Fluorescence Resonance Energy Transfer, U.S. Pat. No.5,661,035 (Aug. 26, 1997); Roger Y. Tsien & Jesus E. Gonzalez, Detectionof Transmembrane Potentials by Optical Methods, U.S. Pat. No. 6,107,066(Aug. 22, 2000).

It is also envisioned that assays involving Fluorescence ResonanceEnergy Transfer (FRET) can be used to detect a change in cell membranepotential. FRET is a distance dependent interaction between theelectronic excited states of two molecules in which excitation istransferred from a donor fluorophore to an acceptor without emission ofa photon. The process of energy transfer results in a reduction(quenching) of fluorescence intensity and excited state lifetime of thedonor fluorophore and, where the acceptor is a fluorophore, can producean increase in the emission intensity of the acceptor. Upon induction ofa persistent Na⁺ current, the membrane is depolarized, resulting aseparation of the donor/acceptor pair and thus the resonance energytransfer is reduced and can be detected, for example, by increased donorfluorescence emission, decreased acceptor fluorescence emission, or by ashift in the emission maxima from near the acceptor emission maxima tonear the donor emission maxima. In the presence of a persistent currentblocker, membrane depolarization and thus changes in FRET are reduced orprevented. If desired, the amount of persistent Na⁺ current reduction orprevention, modulated by a persistent Na⁺ channel, can be calculated asa function of the difference in the degree of FRET using the appropriatestandards.

As a non-limiting example, a FRET pair comprises a voltage-sensitivemobile acceptor DiSBAC²(3) and a fluorescent, membrane-bound donorCC2-DMPE. When the cell interior has a relatively negative potential,the DiSBAC²(3) will bind to the exterior of the cell membrane, resultingin efficient FRET. When the cell interior has a relatively positivepotential, however, the DiSBAC²(3) will bind to the interior of the cellmembrane, thus separating the FRET pair and disrupting FRET. Othernon-limiting examples of fluorophores useful as acceptors for theCC2-DMPE donor are listed in Table 2.

TABLE 2 Donor Fluorophores and Acceptors Donor Acceptor CC2-DMPEDiSBAC²(3) CC2-DMPE DiSBAC⁴(3) CC2-DMPE RH-155 (NK3041) CC2-DMPE RH-479(JPW1131) CC2-DMPE RH-482(JPW1132, NK3630) CC2-DMPE RH-1691 CC2-DMPERH-1692 CC2-DMPE RH-1838 CC2-DMPE R-1114(WW781) CC2-DMPE JPW1177CC2-DMPE JPW1245

Aspects of the present invention provide, in part, a cell. As usedherein, the term “cell,” means any cell that natively expresses themolecules necessary to practice a method disclosed in the presentspecification, such as, e.g., a persistent Na⁺ channel, a transient Na⁺channel a K⁺ channel or a Na/K ATPase pump, or can be geneticallyengineered to express the molecules necessary to practice a methoddisclosed in the present specification, such as, e.g., a persistent Na⁺channel, a transient Na⁺ channel a K⁺ channel or a Na/K ATPase pump. Asa non-limiting example, a cell useful for practicing a method using aNa⁺ depletion/repletion protocol would be a cell that natively express aK⁺ channel, a transient Na⁺ channel and a persistent Na⁺ channel, or acell genetically engineered to express a K⁺ channel, a transient Na⁺channel and a persistent Na⁺ channel. As another non-limiting example, acell useful for practicing a method using a hyperpolarization protocolwould be a cell that natively express a K⁺ channel and a persistent Na⁺channel, or a cell genetically engineered to express a K⁺ channel and apersistent Na⁺ channel. As yet another non-limiting example, a celluseful for practicing a method using a Na/K ATPase pump inhibitorprotocol would be a cell that natively express a persistent Na⁺ channeland Na/K ATPase pump, or a cell genetically engineered to express apersistent Na⁺ channel and Na/K ATPase pump.

A cell can be obtained from a variety of organisms, such as, e.g.,murine, rat, porcine, bovine, equine, primate and human cells; from avariety of cell types such as, e.g., neural and non-neural; and can beisolated from or part of a heterogeneous cell population, tissue ororganism. It is understood that cells useful in aspects of the inventioncan include, without limitation, primary cells; cultured cells;established cells; normal cells; transformed cells; tumor cells;infected cells; proliferating and terminally differentiated cells; andstably or transiently transfected cells. It is further understood thatcells useful in aspects of the invention can be in any state such asproliferating or quiescent; intact or permeabilized such as throughchemical-mediated transfection such as, e.g., calciumphosphate-mediated, diethyl-laminoethyl (DEAE) dextran-mediated,lipid-mediated, polyethyleneimine (PEI)-mediated, polybrene-mediated,and protein delivery agents; physical-mediated transfection, such as,e.g., biolistic particle delivery, microinjection and electroporation;and viral-mediated transfection, such as, e.g., retroviral-mediatedtransfection. It is further understood that cells useful in aspects ofthe invention may include those which express a Na⁺ channel undercontrol of a constitutive, tissue-specific, cell-specific or induciblepromoter element, enhancer element or both.

Naturally occurring cells having persistent sodium current include,without limitation, neuronal cells, such as, e.g., squid axon,cerebellar Purkinje cells, neocortical pyramidal cells, thalamicneurons, CA1 hippocampal pyramidal cells, striatal neurons and mammalianCNS axons. Other naturally occurring cells having persistent sodiumcurrent can be identified by those skilled in the art using methodsdisclosed herein below and other well known methods. Geneticallyengineered cells expressing a persistent Na⁺ current can include,without limitation, isolated mammalian primary cells; establishedmammalian cell lines, such as, e.g., COS, CHO, HeLa, NIH3T3, HEK 293-Tand PC12; amphibian cells, such as, e.g., Xenopus embryos and oocytes;insect cells such as, e.g., D. melanogaster, yeast cells such as, e.g.,S. cerevisiae, S. pombe, or Pichia pastoris and prokaryotic cells, suchas, e.g., E. coli.

Cells can be genetically engineered to express a polynucleotide moleculeencoding a molecule necessary to practice a method disclosed in thepresent specification, such as, e.g., a persistent Na⁺ channel, atransient Na⁺ channel a K⁺ channel or a Na/K ATPase pump. The sequencesof polynucleotide molecules encoding a molecule necessary to practice amethod disclosed in the present specification, such as, e.g., apersistent Na⁺ channel, a transient Na⁺ channel a K⁺ channel or a Na/KATPase pump are well-known and publicly available to one skilled in theart. For example, both polynucleotide and protein sequences of allcurrently described persistent Na⁺ channels, transient Na⁺ channels, K⁺channels and Na/K ATPase pumps are publicly available from the GenBankdatabase (National Institutes of Health, National Library of Medicine.In addition, polynucleotide and protein sequences are described, see,e.g, Alan L. Goldin, Diversity of Mammalian Voltage-gated SodiumChannels, 868 ANN. N.Y. ACAD. SCI. 38-50 (1999), William A. Catterall,From Ionic Currents to Molecular Mechanisms: The Structure and Functionof Voltage-gated Sodium Channels, 26(1) NEURON 13-25 (2000); John N.Wood & Mark D. Baker, Voltage-gated Sodium Channels, 1(1) CURR. OPIN.PHARMACOL. 17-21 (2001); and Frank H. Yu & William A. Catterall,Overview of the Voltage-Gated Sodium Channel Family, 4(3) GENOME BIOL.207 (2003).

Voltage-gated Na⁺ channels are members of a large mammalian gene familyencoding at least nine alpha- (Na_(v)1.1-Na_(v)1.9) and fourbeta-subunits. While all members of this family conduct Na⁺ ions throughthe cell membrane, they differ in tissue localization, regulation and,at least in part, in kinetics of activation and inactivation, see, e.g.,Catterall, supra, (2000); and Sanja D. Novakovic et al., Regulation ofNa ⁺ Channel Distribution in the Nervous System, 24(8) TRENDS NEUROSCI.473-478 (2001). Four sodium channels, Na_(v)1.3, Na_(v)1.5, Na_(v)1.6and Na_(v)1.9, have historically been known to generate a persistentcurrent. Recent evidence, however, suggests that all voltage-gatedsodium channels are capable of producing a persistent current, see,e.g., Abraha Taddese & Bruce P. Bean, Subthreshold Sodium Current fromRapidly Inactivating Sodium Channels Drives Spontaneous Firing ofTubermammillary Neurons, 33(4) NEURON 587-600 (2002); Michael Tri H. Do& Bruce P. Bean, Subthreshold Sodium Currents and Pacemaking ofSubthalamic Neurons: Modulation by Slow Inactivation, 39(1) NEURON109-120 (2003). As of Nov. 21, 2005, accession numbers forrepresentative human voltage-gated Na⁺ channel family members includegi29893559, gi/0337597, gi25014054, gi40255316, gi37622907, gi7657544,gi4506813, gi56748895 and gi7657542, which are hereby incorporated byreference in their entirety.

Voltage-gated K⁺ channels are members of a large mammalian gene familyencoding at least 5 six transmembrane subunits: K_(v)1.x, K_(v)2.x,K_(v)3.x, K_(v)4.x and K_(v)CNQ, see, e.g., Gary Yellen, Thevoltage-gated potassium channels and their relatives, 419(6902) Nature35-42 (2002). These ion channels help maintain and regulate the K⁺-basedcomponent of the membrane potential and are thus central to manycritical physiological processes. Each subunit family is composed ofseveral genes. Thus, the K_(v)1.x family in mammals is comprised of fivedistinct genes: K_(v)1.1, K_(v)1.2, K_(v)1.3, K_(v)1.4 and K_(v)1.5. TheK_(v)2.x family in mammals is comprised of two distinct genes: K_(v)2.1and K_(v)2.2. The K_(v)3.x family in mammals is comprised of fourdistinct genes: K_(v)3.1, K_(v)3.2a, K_(v)3.2b, K_(v)3.2c, K_(v)3.3,K_(v)3.4a and K_(v)3.4b. The K_(v)4.x family in mammals is comprised ofthree distinct genes: K_(v)4.1, K_(v)4.2, K_(v)4.3-1 and K_(v)4.3-2. Asof Nov. 21, 2005, accession numbers for representative humanvoltage-gated K⁺ channel family members include K_(v)1.x channelsgi4557685, gi4826782, gi25952082, gi450-4817 and gi25952087; K_(v)2.xchannels gi4826784 and gi27436974; K_(v)3.x channels gi76825377,gi21217561, gi21217563, gi24497458, gi24497460, gi24497462 andgi24497464; and K_(v)4.x channels gi27436981, gi9789987 and gi27436984,gi27436986, which are hereby incorporated by reference in theirentirety.

The Na/K ATPase pump family is a member of the P-type ATPasesuperfamily. Two subunits α and β comprise the Na/K pump. In mammals,four a isoforms have been identified (α1, α2, α3, α4). A housekeepingfunction is assigned to al. This isoform is expressed throughout thebody. The α2 isoform is expressed mainly in brain, heart and skeletalmuscle and appears to be involved in regulation cell Ca2+. The α4isoform is believed to help maintain sperm motility. The Na/K ATPasepump family in mammals is comprises Na/K α1a, Na/K α1b, Na/K α2, Na/K α3and Na/K α4. As of Nov. 21, 2005, accession numbers for representativehuman Na/K ATPase pump family members include gi21361181, gi48762682,gi1703467, gi29839750 and gi37577153, which are hereby incorporated byreference in their entirety.

Another aspect of the present invention provides, in part, an expressionconstruct that allow for expression of a polynucleotide moleculeencoding a molecule necessary to practice a method disclosed in thepresent specification, such as, e.g., a persistent Na⁺ channel, atransient Na⁺ channel, a K⁺ channel or a Na/K ATPase pump. Theseexpression constructs comprise an open reading frame encoding a moleculenecessary to practice a method disclosed in the present specification,such as, e.g., a persistent Na⁺ channel, a transient Na⁺ channel, a K⁺channel or a Na/K ATPase pump, operably-linked to control sequences froman expression vector useful for expressing a the necessary molecule in acell. The term “operably linked” as used herein, refers to any of avariety of cloning methods that can ligate a polynucleotide moleculedisclosed in the present specification into an expression vector suchthat a polypeptide encoded by the composition is expressed whenintroduced into a cell. Well-established molecular biology techniquesthat may be necessary to make an expression construct disclosed in thepresent specification including, but not limited to, proceduresinvolving polymerase chain reaction (PCR) amplification restrictionenzyme reactions, agarose gel electrophoresis, nucleic acid ligation,bacterial transformation, nucleic acid purification, nucleic acidsequencing are routine procedures well within the scope of one skilledin the art and from the teaching herein. Non-limiting examples ofspecific protocols necessary to make an expression construct aredescribed in e.g., MOLECULAR CLONING A LABORATORY MANUAL, supra, (2001);and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Frederick M. Ausubel et al.,eds. John Wiley & Sons, 2004). These protocols are routine procedureswell within the scope of one skilled in the art and from the teachingherein.

A wide variety of expression vectors can be employed for expressing anopen reading frame encoding a molecule necessary to practice a methoddisclosed in the present specification, such as, e.g., a persistent Na⁺channel, a transient Na⁺ channel, a K⁺ channel or a Na/K ATPase pump,and include without limitation, viral expression vectors, prokaryoticexpression vectors and eukaryotic expression vectors including yeast,insect and mammalian expression vectors. Non-limiting examples ofexpression vectors, along with well-established reagents and conditionsfor making and using an expression construct from such expressionvectors are readily available from commercial vendors that include,without limitation, BD Biosciences-Clontech, Palo Alto, Calif.; BDBiosciences Pharmingen, San Diego, Calif.; Invitrogen, Inc, Carlsbad,Calif.; EMD Biosciences-Novagen, Madison, Wis.; QIAGEN, Inc., Valencia,Calif.; and Stratagene, La Jolla, Calif. The selection, making and useof an appropriate expression vector are routine procedures well withinthe scope of one skilled in the art and from the teachings herein.

It is envisioned that any of a variety of expression systems may beuseful for expressing a construct disclosed in the presentspecification. An expression system encompasses both cell-based systemsand cell-free expression systems. Cell-based systems include, withoutlimited, viral expression systems, prokaryotic expression systems, yeastexpression systems, baculoviral expression systems, insect expressionsystems and mammalian expression systems. Cell-free systems include,without limitation, wheat germ extracts, rabbit reticulocyte extractsand E. coli extracts. Expression using an expression system can includeany of a variety of characteristics including, without limitation,inducible expression, non-inducible expression, constitutive expression,viral-mediated expression, stably-integrated expression, and transientexpression. Expression systems that include well-characterized vectors,reagents, conditions and cells are well-established and are readilyavailable from commercial vendors that include, without limitation,Ambion, Inc. Austin, Tex.; BD Biosciences-Clontech, Palo Alto, Calif.;BD Biosciences Pharmingen, San Diego, Calif.; Invitrogen, Inc, Carlsbad,Calif.; QIAGEN, Inc., Valencia, Calif.; Roche Applied Science,Indianapolis, Ind.; and Stratagene, La Jolla, Calif. Non-limitingexamples on the selection and use of appropriate heterologous expressionsystems are described in e.g., PROTEIN EXPRESSION. A PRACTICAL APPROACH(S. J. Higgins and B. David Hames eds., Oxford University Press, 1999);Joseph M. Fernandez & James P. Hoeffler, GENE EXPRESSION SYSTEMS. USINGNATURE FOR THE ART OF EXPRESSION (Academic Press, 1999); and Meena Rai &Harish Padh, Expression Systems for Production of Heterologous Proteins,80(9) CURRENT SCIENCE 1121-1128, (2001). These protocols are routineprocedures well within the scope of one skilled in the art and from theteaching herein.

An expression construct comprising a polynucleotide molecule encoding amolecule necessary to practice a method disclosed in the presentspecification, such as, e.g., a persistent Na⁺ channel, a transient Na⁺channel, a K⁺ channel or a Na/K ATPase pump, can be operationally-linkedto a variety of regulatory elements that can positively or negativelymodulate, either directly or indirectly, the expression of apolynucleotide molecule, such as, e.g., constitutive, tissue-specific,inducible or synthetic promoters and enhancers. Using such systems, oneskilled in the art can express the desired levels of a moleculenecessary to practice a method disclosed in the present specification,such as, e.g., a persistent Na⁺ channel, a transient Na⁺ channel, a K⁺channel or a Na/K ATPase pump, using routine laboratory methods asdescribed, see, e.g., Molecular Cloning A Laboratory Manual (JosephSambrook & David W. Russell eds., Cold Spring Harbor Laboratory Press,3^(rd) ed. 2001); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FrederickM. Ausubel et al., eds., John Wiley & Sons, 2004). Non-limiting examplesof constitutive regulatory elements include, e.g., the cytomegalovirus(CMV), herpes simplex virus thymidine kinase (HSV TK), simian virus 40(SV40) early, 5′ long terminal repeat (LTR), elongation factor-1a(EF-1α) and polybiquitin (UbC) regulatory elements. Non-limitingexamples of inducible regulatory elements useful in aspects of thepresent invention include, e.g., chemical-inducible regulatory elementssuch as, without limitation, alcohol-regulated, tetracycline-regulated,steroid-regulated, metal-regulated and pathogenesis-related; andphysical-inducible regulatory elements such as, without limitation,temperature-regulated and light-regulated. Such inducible regulatoryelements can be prepared and used by standard methods and arecommercially available, including, without limitation,tetracycline-inducible and tetracycline-repressible elements such as,e.g., Tet-On™ and Tet-Off™ (BD Biosciences-Clontech, Palo Alto, Calif.)and the T-REx™ (Tetracycline-Regulated Expression) and Flp-In™ T-REx™systems (Invitrogen, Inc., Carlsbad, Calif.); ecdysone-inducibleregulatory elements such as, e.g., the Complete Controle® InducibleMammalian Expression System (Stratagene, Inc., La Jolla, Calif.);isopropyl β-D-galactopyranoside (IPTG)-inducible regulatory elementssuch as, e.g., the LacSwitch®^(II) Inducible Mammalian Expression System(Stratagene, Inc., La Jolla, Calif.); and steroid-inducible regulatoryelements such as, e.g., the chimeric progesterone receptor induciblesystem, GeneSwitch™ (Invitrogen, Inc., Carlsbad, Calif.). The skilledperson understands that these and a variety of other constitutive andinducible regulatory systems are commercially available or well known inthe art and can be useful in the invention for controlling expression ofa polynucleotide which encodes a molecule necessary to practice a methoddisclosed in the present specification, such as, e.g., a persistent Na⁺channel, a transient Na⁺ channel, a K⁺ channel or a Na/K ATPase pump.

Aspects of the present invention provide, in part, cells comprising acertain gK/gNa ratio, or relative gK/gNa conductance. The relativegK/gNa conductance of genetically engineered cells can be measuredsimply by increasing the extracelluar K⁺ concentration and monitoringthe change in membrane potential. Contributions of other ions, such as,e.g., Cl⁻, to the overall membrane potential can be controlled bysubstituting non-permeant analogs or pharmacological blockers to preventtheir contribution to the equilibrium potential. The relative gK/gNaconductance can be calculated using the modified form of the ChordConductance equation below:

ΔE _(m)=λ(E _(K2) −E _(K1))/(λ+1)

Where: λ=GK/GNa; EK₂=the equilibrium potential for K⁺ following a10-fold increase in extracellular K⁺; EK₁=the equilibrium potential forK prior to increasing K⁺10-fold.

For any ion the equilibrium potential is defined as

E _(i)=(RT/ZF)log [I _(out) /I _(in)]

Where: at physiological temperature, with monovalent ions, RT/ZF=60 mV;and I_(out) and I_(in) are the concentrations of the relevant ion in theextracellular and intracellular compartments respectively.

As a non-limiting example, to determine the relative gK/gNa conductancefor a particular cell line, a 10-fold increase in extracellular K⁺concentration is added to the physiological solution. This added K⁺induces a depolarization by shifting the equilibrium potential byapproximately 60 mV in the positive direction. The values obtained fromthe experiment can then be used in the modified form of the ChordConductance equation above to calculate the relative gK/gNa conductance.For a Na/K ATPase pump inhibitor protocol, a relative gK/gNa conductance≧20-fold is indicative of a depolarization near the theoreticalequilibrium potential 60 mV, and thus suitable for this protocol.

Aspects of the present invention provide, in part, detectingfluorescence emitted by the voltage-sensitive dye. The fluorescenceemitted from a sample is typically determined using a fluorimeter. Influorescence detection relying on a single fluorophore, an excitationradiation from an excitation source passes through excitation optics andexcites the voltage-sensitive dye. In response, voltage-sensitive dyeemits radiation which has an emission wavelength that is different fromthe excitation wavelength, which is collected by collection optics. Influorescence detection relying on FRET, an excitation radiation from anexcitation source having a first wavelength passes through excitationoptics. The excitation optics cause the excitation radiation to excitethe voltage-sensitive dye. In response, voltage-sensitive dye emitsradiation which has an emission wavelength that is different from theexcitation wavelength, which is collected by collection optics. Ifdesired, the device includes a temperature controller to maintain thecell at a specific temperature while being scanned. If desired, a multiaxis translation stage moves a microtiter plate containing a pluralityof samples in order to position different wells to be exposed. It isunderstood that the multi-axis translation stage, temperaturecontroller, auto-focusing feature, and electronics associated withimaging and data collection can be managed by the appropriate digitalcomputer.

It is further understood that the methods of the invention can beautomated and can be configured in a high throughput or ultrahigh-throughput format using, without limitation, 96 well, 384-well or1536 well plates. Instrumentation useful for measuring membranepotential for high-throughput screening procedures include, withoutlimitation, Fluorometric Imaging Plate Reader (FLIPR®; MolecularDevices, Sunnyvale, Calif.) and Voltage/Ion Probe Reader (VIPR; AuroraBiosciences, San Diego, Calif.), see, e.g., Roger Y. Tsien & Jesus E.Gonzalez, Detection of Transmembrane Potentials by Optical Methods, U.S.Pat. No. 6,342,379 (Jan. 29, 2002); Jesus E. Gonzalez & Michael P.Maher, Cellular Fluorescent Indicators and Voltage/Ion Probe Reader(VIPR) Tools for Ion Channel and Receptor Drug Discovery, 8(5-6)RECEPTORS CHANNELS 283-295, (2002); and Michael P. Maher & Jesus E.Gonzalez, High Throughput Method and System for Screening CandidateCompounds for Activity Against Target Ion Channels, U.S. Pat. No.6,686,193 (Feb. 3, 2004). As a non-limiting example, fluorescenceemission can be detected using the FLIPR® instrumentation system, whichis designed for 96-well plate assays. FLIPR® utilizes a water-cooled 488nm argon ion laser (5 watt) or a xenon arc lamp and a semiconfocaloptical system with a charge coupled device (CCD) camera to illuminateand image the entire plate. The FPM-2 96-well plate reader (FolleyConsulting and Research; Round Lake, Ill.) also can be useful indetecting fluorescence emission in the methods of the invention. Oneskilled in the art understands that these and other automated systemswith the appropriate spectroscopic compatibility can be useful inhigh-throughput screening methods disclosed in the presentspecification.

Aspects of the present invention provide, in part, determining arelative emitted fluorescence. A relative emitted fluorescence isdetermined by comparing the fluorescence emitted from a test sample tothe corresponding control sample for that test sample. A decrease inemitted fluorescence from a test sample relative to a control sample isindicative of a reduction or prevention of a persistent Na⁺ current,i.e., the presence of a persistent Na⁺ channel blocker in the testsample. As a non-limiting example, a decrease in emitted fluorescencefrom a test sample relative to a control sample using a Na⁺depletion/repletion protocol is indicative of a reduction or preventionof a persistent Na⁺ current, i.e., the presence of a persistent Na⁺channel blocker in the test sample. As another non-limiting example, adecrease in emitted fluorescence from a test sample relative to acontrol sample using a hyperpolarization protocol is indicative of areduction or prevention of a persistent Na⁺ current, i.e., the presenceof a persistent Na⁺ channel blocker in the test sample. As yet anothernon-limiting example, a decrease in emitted fluorescence from a testsample relative to a control sample using a Na/K ATPase pump inhibitorprotocol is indicative of a reduction or prevention of a persistent Na⁺current, i.e., the presence of a persistent Na⁺ channel blocker in thetest sample.

In an embodiment, a decrease in emitted fluorescence from a test samplerelative to a control sample is indicative of a reduction or preventionof a persistent Na⁺ current, i.e., the presence of a persistent Na⁺channel blocker. In aspects of this embodiment, a decreased relativeemitted fluorescence from a test sample can be, e.g., at least two-fold,at least three-fold, at least four-fold, at least five-fold, at leastten-fold, at least twenty-fold or more as compared to the relativeemitted fluorescence from a control sample. In other aspects of thisembodiment, a decreased relative emitted fluorescence from a test samplecan be, e.g., at most two-fold, at most three-fold, at most four-fold,at most five-fold, at most ten-fold, at most twenty-fold as compared tothe relative emitted fluorescence from a control sample.

In another embodiment, an increase is emitted fluorescence from acontrol sample relative to a test sample is indicative of a reduction orprevention of a persistent Na⁺ current, i.e., the presence of apersistent Na⁺ channel blocker. In aspects of this embodiment, anincreased relative emitted fluorescence from a control sample can be,e.g., at least two-fold, at least three-fold, at least four-fold, atleast five-fold, at least ten-fold, at least twenty-fold or more ascompared to the relative emitted fluorescence from a test sample. Inother aspects of this embodiment, an increased relative emittedfluorescence from a control sample can be, e.g., at most two-fold, atmost three-fold, at most four-fold, at most five-fold, at most ten-fold,at most twenty-fold as compared to the relative emitted fluorescencefrom a test sample.

Aspects of the present invention provide a method for identifying aselective blocker of a persistent Na⁺ channel using a Na⁺depletion/repletion protocol (FIG. 2). This protocol relies on theessential requirement of Na⁺ to generate a persistent current.Generally, cells having a K⁺ channel, a transient Na⁺ channel and apersistent Na⁺ channel are incubated in a Na⁺-free physiologicalsolution. A small depolarization of the membrane of the cells is inducedto activate both transient and persistent Na⁺ channels. However, noadditional depolarization will be observed in the absence ofextracellular Na⁺. Thus, within a few milliseconds followingdepolarization the transient Na⁺ channels will close, but the channelscapable of generating a persistent Na⁺ current will remain open. Theaddition of Na⁺ to the Na⁺-free physiological solution will cause theseopened persistent Na⁺ channels to generate a persistent current causingthe membrane to depolarize. The presence of a persistent Na⁺ channelblocker will either eliminate or reduce the magnitude of thisdepolarization event (FIG. 3). Therefore, screens based on the Na⁺depletion/repletion protocol can identify a potential persistent Na⁺channel blocker by absent or reduction of membrane depolarization.Conversely, molecules that lack this blocking capability, whether apotential test molecule or a control sample, will not affect themagnitude of this depolarization event. The Na⁺ depletion/repletionprotocol therefore allows the discovery of molecules that block thepersistent Na⁺ current and as such is a screen for persistent Na⁺channels blockers.

In one embodiment, a Na⁺ depletion/repletion protocol test samplecomprises a cell comprising a K⁺ channel, a transient Na⁺ channel and apersistent Na⁺ channel.

Aspects of the present invention provide, in part, depolarizing amembrane of the cell. A cell membrane may be depolarized by adding K⁺ tothe medium to shift the K⁺ equilibrium potential in the positivedirection. As an non-limiting example, in a cell in which the K⁺conductance dominates at the resting potential and the intracellular andextracellular K⁺ concentrations are 120 mM and 4.5 mM respectively theequilibrium potential for potassium would be approximately −84 mV.Addition of K⁺ to bring the extracellular K⁺ to 13 mM would result in aequilibrium potential for potassium of approximately −57 mV. Dependingon the relative contribution of the other ionic conductances of the cellmembrane this increase in K⁺ could result in depolarization of up to 27mV. One skilled in the art will recognize that there are many othermethods for depolarizing a cell membrane. As additional non-limitingexamples it would be possible to depolarize the cell membrane by addingK⁺ channel blockers such as the ions, e.g., Cs⁺, Ba²⁺, TEA⁺, or smallorganic molecules, e.g., 4-aminopyridine, quinidine or phencyclidine, orpeptide toxins e.g. charybdotoxin, margatoxin, iberiotoxin, noxiustoxin,kaliotoxin to the extracellular medium. One skilled in the art wouldrecognize that inhibition of the electrogenic Na⁺/K⁺ pump with cardiacglycosides such as ouabain, or and dihydro-ouabain; isothiouronium orderivative thereof, such as, e.g., 1-bromo-2,4,6-tris(methylisothiouronium) benzene (Br-TITU) and 1,3-dibromo-2,4,6-tris(methylisothiouronium) benzene (Br2-TITU); digitoxigenin or derivativethereof, such as, e.g., digitalis, 22-benzoyloxy-digitoxigenin,22-acetoxy-digitoxigenin, 22-allyl-digitoxigenin,22-hydroxy-digitoxigenin and 14β, 17β-cycloketoester-3β-OH androstane(INCICH-D7); coumestan or derivative thereof, such as, e.g.,2-methoxy-3,8,9-trihydroxy coumestan (PCALC36); vanadate or derivativethereof; cardenolide or derivative thereof; and natural cardiacglycosides would depolarize the cell membrane. Additionally, one skilledin the art would recognize that the use of electric field stimulation(EFS) to deliver electrical stimuli to the cell would result in adepolarization of the cell membrane. Each of the above non-limitingexamples could be used in combination with each other or other methodsto deliver depolarizing stimuli to the cell membrane.

In one embodiment, depolarizing a membrane of the cell can be with theaddition of K⁺. It is envisioned that any K⁺ concentration can be usefulwith the proviso that this K⁺ addition induces a membrane depolarizationof at least 5 mV and such addition does not prevent the additionaldepolarization due to Na⁺ repletion. For example, a K⁺-induceddepolarization of range of about 5 to about 50 mV. In aspects of thisembodiment, the K⁺-induced depolarization can be, e.g., about 5 mV,about 10 mV, about 20 mV, about 30 mV, about 40 mV or about 50 mV.

Aspects of the present invention provide, in part, generating a currentthrough the persistent Na⁺ channel by adding a Na⁺ containing solutioninto a well containing cells depolarized in the absence Na⁺. Themagnitude of the depolarization will depend on the concentration of Na⁺added and the relative conductance of the Na⁺ channels generating thepersistent current. As a non-limiting example addition of Na⁺ to theextracellular solution that results in a final Na⁺ concentration of70-100 mM will result in a robust depolarization of the cell membrane inthe presence of persistent sodium channels. In addition, one skilled inthe art will recognize that to obtain a reliable measurement ofpersistent Na⁺ current a wide range timings for the applications of theNa⁺ solution would be possible as long as the transient sodium channelswere allowed to inactivate.

Aspects of the present invention provide a method for identifying aselective blocker of a persistent Na⁺ channel using a hyperpolarizationprotocol (FIG. 4). In this protocol, the proportion of K⁺ and persistentNa⁺ channels present in cells is such that their conductances areessentially equal. Assuming all other ion conductances are minimal theresting membrane potential will lie approximately halfway between theequilibrium potential for Na⁺ and the equilibrium potential of K⁺. Underthese conditions adding a K channel blocker will depolarize the cellstoward the equilibrium potential of Na⁺ (ENa>50 mV). On the other hand,adding a persistent Na⁺ channel blocker will hyperpolarize the cellsdriving the membrane potential towards the equilibrium potential for K⁺(EK<-85 mV). These predictions can be understood via the chordconductance equation when a cell is solely permeable to Na⁺ and K⁺ (themedia is Cl⁻-free and appropriate inhibitors are present as in the Na⁺depletion/repletion protocol. Thus, the presence of a persistent Na⁺channel blocker generates a hyperpolarization event (FIG. 5). The morepotent the persistent Na⁺ channel blocker the greater thehyperpolarization with complete block bringing the membrane potential toEK. Therefore, screens based on the hyperpolarization protocol canidentify a potential persistent Na⁺ channel blocker by the induction ofmembrane hyperpolarization. Conversely, molecules that lack thisblocking capability, whether a potential test molecule or a controlsample, will not induce this hyperpolarization event. Thehyperpolarization protocol therefore allows the discovery of moleculesthat block the persistent Na⁺ current and as such is a screen forpersistent Na⁺ channels blockers.

In an embodiment, a hyperpolarization protocol test sample comprises acell comprising a K⁺ channel and a persistent Na⁺ channel wherein aresting membrane potential of the cell is approximately halfway betweenthe equilibrium potential of K⁺ and the equilibrium potential of Na⁺. Inaspects of this embodiment, the resting membrane potential can comprisean approximate range of, e.g., −50 mV to 15 mV, −45 mV to 10 mV, −40 mVto 5 mV, −35 mV to 0 mV, −30 mV to −5 mV, −25 mV to −10 mV or −20 mV to−15 mV. In other aspects of this embodiment, the resting membranepotential can comprise an approximate range of, e.g., −50 mV to −20 mV,−40 mV to −10 mV, −30 mV to 0 mV, −20 mV to 10 mV or −mV to 20 mV.

In another embodiment, a hyperpolarization protocol test samplecomprises a cell comprising a K⁺ channel and a persistent Na⁺ channelwherein a resting membrane potential of the cell is approximatelyhalfway between the equilibrium potential of K⁺ and the equilibriumpotential of Na⁺ can detect a hyperpolarization of a membrane. Inaspects of this embodiment, membrane potential can hyperpolarize by atleast 20 mV, at least 30 mV, at least 40 mV, at least 50 mV or at least60 mV. In other aspects of this embodiment, membrane potential canhyperpolarize by at most 20 mV, at most 30 mV, at most 40 mV, at most 50mV or at most 60 mV.

Aspects of the present invention provide a method for identifying aselective blocker of a persistent Na⁺ channel using a Na/K ATPase pumpinhibitor protocol, see FIG. 6. This assay relies on the fact thatinhibition of the Na/K ATPase will allow net cellular Na⁺ entry and Kloss. In this protocol cells containing persistent Na⁺ channels, K⁺channels, and Na/K ATPase, assayed in a Cl⁻-free medium physiologicalsolution, are treated with a pump inhibitor. This inhibition will leadto an initial small membrane depolarization due to blockage of the Na/KATPase pump and a subsequent large secondary depolarization. Thissecondary depolarization is the key to the assay and relies on the factthat the equilibrium potential for K⁺ will become more positive as thecells lose K⁺. The rationale is as follows. For this assay to work GKmust be >>GNa_(persistent). Following addition of a Na/K ATPase pumpinhibitor, the cells will gain Na⁺ via persistent Na⁺ channels that areopen at near resting membrane potential. In the absence of a Clconductance the Na⁺ gained by the cells will be electrically compensatedfor by an equimolar loss of K⁺. Since GK>>GNa, the membrane potentialwill be dominated by K and therefore a decrease in cell K⁺ will resultin a positive change in the potassium equilibrium potential. As aresult, a depolarization of the membrane will occur because ofmillimolar K⁺ loss. It should be understood that although the cell gainsNa⁺ this gain is of little effect on the membrane potential sinceGK>>GNa. Instead it is the compensatory movement of K⁺ ions drives themembrane potential in this assay. Thus, the extent of the depolarizationwill depend on the amount K⁺ lost by the cell following the addition ofa Na/K ATPase pump inhibitor. The presence of a persistent Na⁺ channelblocker will either eliminate or reduce the magnitude of this secondarydepolarization event (FIG. 7). Therefore, screens based on the Na/KATPase pump inhibitor protocol can identify a potential persistent Na⁺channel blocker by absent or reduction of the secondary depolarizationof the membrane. Conversely, molecules that lack this blockingcapability, whether a potential test molecule or a control sample, willnot affect the magnitude of this depolarization event. The Na/K ATPasepump inhibitor protocol therefore allows the discovery of molecules thatblock the persistent Na⁺ current and as such is a screen for persistentNa⁺ channels blockers.

In an embodiment, a Na/K ATPase pump inhibitor protocol test samplecomprises a cell comprising a K⁺ channel and a persistent Na⁺ channelwherein a K⁺ conductance of the K⁺ channel is greater than the Na⁺conductance from the persistent Na⁺ channel. In aspect of thisembodiment, the K⁺ conductance of the K⁺ channel is greater than the Na⁺conductance by, e.g., at least 10-fold higher, at least 20-fold higher,at least 30-fold higher, at least 40-fold higher, at least 50-fold Inother aspect of this embodiment, the K⁺ conductance of the K⁺ channel isgreater than the Na⁺ conductance by, e.g., at most 10-fold higher, atmost 20-fold higher, at most 30-fold higher, at most 40-fold higher, atmost 50-fold higher or at most 60-fold higher.

Aspects of the present invention provide, in part, a Na/K ATPase pumpinhibitor. It is envisioned that any molecule capable of inhibiting theactivity of a Na/K ATPase pump can be useful. Non-limiting examples of aNa/K ATPase pump inhibitor include oubain or derivative thereof, suchas, e.g., ouabain and dihydro-ouabain; isothiouronium or derivativethereof, such as, e.g., 1-bromo-2,4,6-tris (methylisothiouronium)benzene (Br-TITU) and 1,3-dibromo-2,4,6-tris (methylisothiouronium)benzene (Br2-TITU); digitoxigenin or derivative thereof, such as, e.g.,digitalis, 22-benzoyloxy-digitoxigenin, 22-acetoxy-digitoxigenin,22-allyl-digitoxigenin, 22-hydroxy-digitoxigenin and 14β,17β-cycloketoester-3β-OH androstane (INCICH-D7); coumestan or derivativethereof, such as, e.g., 2-methoxy-3,8,9-trihydroxy coumestan (PCALC36);vanadate or derivative thereof; cardenolide or derivative thereof; andnatural cardiac glycosides. The magnitude of the depolarization willdepend on the concentration of inhibitor added and the absoluteconductance of the Na⁺ channels generating the persistent current.

In another embodiment, depolarizing a membrane of the cell can be with aNa/K ATPase pump inhibitor. In an embodiment, a Na/K ATPase pumpinhibitor depolarizes the cell membrane by inhibiting Na/K ATPase pumpactivity. In aspects of this embodiment, a Na/K ATPase pump inhibitorused can be, e.g., oubain or derivative thereof, an isothiouronium orderivative thereof, digitoxigenin or derivative thereof, a coumestan orderivative thereof, vanadate or derivative thereof, a cardenolide orderivative thereof or a natural cardiac glycoside.

Aspects of the present invention provide a method for identifying aselective blocker of a persistent Na⁺ channel using transient Na⁺current protocol, see FIG. 8. Protocols such as, e.g., a Na⁺depletion/repletion protocol, a hyperpolarization protocol and a Na/KATPase pump inhibitor protocol, can allow for the identification of amolecule that reduces or prevents a persistent Na⁺ current. However,these protocols do not address whether the persistent Na⁺ channelblockers found selectively block persistent Na⁺ channels, or also blockNa⁺ channels generating the transient current. Thus another part of thescreen in accordance with aspects of the present invention addresses howmolecules that selectively block persistent Na⁺ current but nottransient Na⁺ current can be distinguished, i.e., identification of aselective persistent Na⁺ channel blocker (FIG. 9). In general, apersistent Na⁺ channel blocker, as determined from a persistent Na⁺channel assay, such as, e.g., a Na⁺ depletion/repletion protocol, ahyperpolarization protocol and a Na/K ATPase pump inhibitor protocol, isretested for its ability to block a transient current. A persistent Na⁺channel blocker that is selective for a persistent Na⁺ channel will notgreatly affect transient Na⁺ current. On the other hand, a persistentNa⁺ channel blocker that reduces or prevents transient Na⁺ current aswell would not be considered a selective persistent Na⁺ channel blocker.

It is envisioned that any and all protocols useful for determining atransient Na⁺ current can be used, including, without limitation, fieldstimulation. In a field stimulation protocol, electrodes are placed inthe well and generate a stimulating current through the cell sufficientto generate an action potential before and after the addition of thepersistent Na⁺ channel blocker. The use of EFS to activate ion channelsis a standard procedure well known to one skilled in the art, see, e.g.,J. Malmivuo and R. Plonsey, Bioelectromagnetism: Principles andApplications of Bioelectric and Biomagnetic Fields, (Oxford UniversityPress, New York. 1-472 pp. 1995); and J. P. Reilly, ElectricalStimulation and Electropathology (Cambridge University Press, Cambridge.1-522 pp, 1992). Furthermore, methods to implement these protocols inHTS format have been described, see, e.g., Michael P. Maher & Jesus E.Gonzalez, Multi-well Plate and Electrode Assemblies for Ion ChannelAssays, U.S. Pat. No. 6,969,449 (Nov. 29, 2005); and Paul Burnett etal., Fluorescence Imaging of Electrically Stimulated Cells, 8(6) J.Biomol. Screen. 660-667 (2003).

In an embodiment, a field stimulation protocol can depolarize a membraneof the cell.

Aspects of the present invention provide, in part, comparing the emittedfluorescence. Comparisons of emitted fluorescence is achieved bycomparing the emitted fluorescence from a persistent Na⁺ current assayrelative to an emitted fluorescence from a transient Na⁺ current assayfor the same potential persistent Na⁺ channel blocker. With respect tothe Na⁺ depletion/repletion, hyperpolarization and Na/K ATPase pumpinhibitor protocols, a decrease is emitted fluorescence from apersistent Na⁺ current assay relative to a transient Na⁺ current assayis indicative of a selective reduction or prevention of a persistent Na⁺current relative to a transient Na⁺ current, i.e., the presence of aselective persistent Na⁺ channel blocker in the test sample.

Although there has been hereinabove described a method and screen foridentifying a Na⁺ channel blocker, in accordance with the presentinvention, for the purposes of illustrating the manner in which theinvention may be used to advantage, it will be appreciated that theinvention is not limited thereto. Accordingly, any and all modification,variations or equivalent arrangements which may occur to those skilledin the art should be considered to be within the scope of the inventionas defined in the appended claims.

EXAMPLES Example 1 Screening Assay for Identifying Persistent SodiumCurrent Blockers using FRET Technology

To establish an assay plate, HEK-293 cells grown in Minimum EssentialMedium (Invitrogen, Inc., Carlsbad, Calif.) supplemented with 10% FetalBovine Serum (Invitrogen, Inc., Carlsbad, Calif.), 1%Penicillin-Streptomycin (Invitrogen, Inc., Carlsbad, Calif.) were eithertransiently or stably transfected with a polynucleotide moleculeexpressing a Na_(v)1.3 sodium channel capable of mediating persistentsodium current. Stably transfected cells were grown in the presence of500 mg/mL G418 Geneticin (Invitrogen, Inc., Carlsbad, Calif.) and 2 μMTTX (Calbiochem, Inc., San Diego, Calif.) to maintain selectivepressure. Cells were grown in vented cap flasks, in 90% humidity and 10%CO₂, to about 80% confluence, harvested by trypsinization and celldensity was determined. Approximately 16 to 24 hours before the assay,each well of a clear-bottom, black-wall 96-well plate (Becton-Dickinson,San Diego, Calif.) coated with Matrigel (Becton-Dickinson, San Diego,Calif.) was seeded with approximately 75,000 HEK-Na_(v)1.3 cells in 150μL of supplemented MEM. Cells were sometimes incubated in 96-well platesat somewhat lower densities (20,000 per well), and incubated for up to40-48 hours.

To examine the ability of test molecules to alter persistent sodiumcurrent, the medium was aspirated, HEK cells were washed 3 times with150 uL of TEA-MeSO₃ solution using CellWash (Thermo LabSystems,Franklin, Mass.) and 150 uL of a Na⁺-free media and physiologicconcentrations of K⁺ (4.5 mM) was added. Extracellular Cl was replacedwith MeSO₃ during preincubation and throughout the assay. Thiseliminates a complicating Cl current during the assay and results in anamplified and more stable voltage-change induced by the persistent Na⁺current. The HEK cells were preincubated for 30-60 minutes with theion-sensitive FRET dye CC2-DMPE (final concentration 10 μM). CC2-DMPE isa stationary coumarin-tagged phospholipid resonance energy donor thathas an optimal excitation wavelength at approximately 405 nm wavelengthlight and an optimal emission wavelength at approximately at 460 nm.While the HEK cells were being stained with coumarin, a 10 μM DiSBAC₂(3)solution in TEA-MeSO₃ solution was prepared. DiSBAC₂(3) is a mobileresonance energy acceptor that partition across the membrane as afunction of the electric field. The optimal excitation spectra for thesedyes overlap the emission spectra of the coumarin donor and, thus, theyact as FRET acceptors. DiSBAC₂(3) has an emission spectrum in the rangeof 570 nm. In addition to DiSBAC₂(3), this solution contained any testmolecule being tested or a DMSO control, at 4 times the desired finalconcentration (e.g., 20 μM for 5 μM final), 1.0 mM ESS-AY17 to reducebackground fluorescence, and 400 μM CdCl₂, which stabilizes the membranepotential of the cells at negative resting potential, resulting in themaximum number of Na⁺ channels being available for activation. After30-60 minutes of CC2-DMPE staining, the cells were washed 3 times with150 μL of TEA-MeSO₃ solution. Upon removing the solution, the cells wereloaded with 80 μL of the DiSBAC₂(3) solution and incubated for 20-30minutes as before. Typically, wells in one column on each plate werefree of test drug(s) and served as positive and negative controls.

The assay plates were then transferred to a voltage/ion probe reader(VIPR) (Aurora Biosciences, San Diego, Calif.) and the VIPR was adjustedso that the fluorescent emission ratio from the donor ands acceptor FRETdyes equaled 1.0. To elicit persistent sodium current, a double additionprotocol was performed by first adding 240 μL of NaMeSO₃ solution toadjust the concentration of sodium and potassium ions in the well to 110mM and 10 mM, respectively, and measuring the resulting sodium-dependentdepolarization and second by adding K⁺ to a final concentration of 80mM, and measuring potassium-dependent depolarization. 240 μL ofTEA-MeSO₃ solution or 1 μM TTX was used as a positive control. Testcompounds that block the Na⁺-dependent signal, but not the K⁺-dependentsignal were selected for further analysis. The Na⁺-dependentdepolarization resulting from the persistent Na⁺ was measured as shownin FIG. 10. The labeled boxes indicate the application of Na⁺ or K⁺.Circles indicate the control response with 0.1% DMSO added, trianglesshow the effects of the Na⁺ channel inhibitor tetracaine (10 μM), andthe diamonds show the response during the application of a non-specificchannel blocker.

In this high-throughput assay, non-specific blockers that inhibitmembrane depolarization induced by any effector must be distinguishedfrom selective persistent Na⁺ current blockers, which block onlypersistent Na⁺-dependent depolarizations. Therefore, a counter-screen todetermine the ability of compounds to alter K⁺-dependent depolarizationwas performed. As shown in FIG. 10, following pre-incubation withvehicle alone (DMSO) both Na⁺ and K⁺ additions produced a robustdepolarization as indicated by the increase in Rf/Ri. Tetracaine, a Na⁺channel blocker, inhibited the Na⁺-dependent, but not the K⁺-dependentchange in Rf/Ri. In contrast, a non-specific inhibitor of Na⁺ andK⁺-dependent depolarization blocked the change in Rf/Ri following eitheraddition. This data demonstrates that selective blockers of thepersistent sodium current can be identified using the described method.

To eliminate compounds that non-specifically inhibited the Na⁺-dependentdepolarization, data obtained using the above procedure were analyzedwith respect to a counter-screen that used K⁺-dependent depolarizationas a readout. To select hits from the primary screen, the data wereplotted as histograms. Inhibition of the Na⁺-dependent depolarizationwas plotted against inhibition of the K⁺-dependent depolarization. Basedon these data, the criteria for selection as a hit, was a greater orequal to 90% inhibition of the Na⁺-dependent depolarization and a lessthan or equal to 20% inhibition of the K⁺-dependent depolarization. Thisprotocol provided a distinction between compounds that were inert ornon-specific in their effects and compounds that specifically block thepersistent sodium current.

Optical experiments in microtiter plates were performed on theVoltage/Ion Probe Reader (VIPR) using two 400 nm excitation filters andfilter sticks with 460 nm and 570 nm filters on the emission side forthe blue and red sensitive PMTs, respectively. The instrument was run incolumn acquisition mode with 2 or 5 Hz sampling and 30 seconds ofrecording per column. Starting volumes in each well were 80 mL; usually240 mL was added to each well during the course of the experiment. Thelamp was allowed to warm up for about 20 minutes and power to the PMTswas turned on for about 10 minutes prior to each experiment.

Ratiometric measurements of changes in fluorescent emissions at 460- and570 nm on the VIPR platform (Aurora Bioscience, San Diego, Calif.)demonstrated that this assay format produces a robust and reproduciblefluorescent signal upon depolarization of HEK-Na_(v)1.3 cells with aNa⁺/K⁺ addition. From a normalized ratio of 1.0 in Na⁺-free media,Na⁺-dependent depolarization resulted in an increase in the 460/570ratio to over 2.2 (FIG. 10). Inter-well analysis of the ratios indicatedthat the amplitude of signal was large enough and consistent enough tobe used in high-throughput screening.

Data were analyzed and reported as normalized ratios of intensitiesmeasured in the 460 nm and 580 nm channels. The VIPR sampling ratevaried between 2 and 5 Hz in different experiments, with 5 Hz used forhigher resolution of the peak sodium responses. The process ofcalculating these ratios was performed as follows. On all plates, column12 contained TEA-MeSO₃ solution with the same DiSBAC2(3) and ESS-AY17concentrations as used in the cell plates; however no cells wereincluded in column 12. Intensity values at each wavelength were averagedfor the duration of the scan. These average values were subtracted fromintensity values in all assay wells. The initial ratio obtained fromsamples 5-10 (Ri) was defined as:

${Ri} = \frac{{Intensity}_{{460\mspace{11mu} {nm}},{{samples}\mspace{11mu} 5\text{-}10}} - {background}_{460\mspace{11mu} {nm}}}{{Intensity}_{{580\mspace{11mu} {nm}},{{samples}\mspace{11mu} 5\text{-}10}} - {background}_{580\mspace{11mu} {nm}}}$

and the ratio obtained from sample f (Rf) was defined as:

${Rf} = \frac{{Intensity}_{{460\mspace{11mu} {nm}},{{sample}\mspace{11mu} f}} - {background}_{460\mspace{11mu} {nm}}}{{Intensity}_{{580\mspace{11mu} {nm}},{{sample}\mspace{11mu} f}} - {background}_{580\mspace{11mu} {nm}}}$

Final data were normalized to the starting ratio of each well andreported as Rf/Ri. The fluorescent response in the Na_(v)1.3 persistentcurrent assay reached a peak approximately 10 seconds following thestart of the run, therefore, the maximum ratio was selected as thereadout for the assay (FIG. 10).

The assay format described above allows for quality assurance bymeasuring both negative (DMSO 0.1%) and positive (tetracaine 10 μM)controls. Every 10th plate in an assay run was a control plate. The datafrom these plates were used to verify that the assay conditions wereoptimal and to normalize the data from the test compounds. FIG. 11 showsresults from control plates from multiple assays.

In FIG. 11, control plates having wells containing either 0.1% DMSO or10 μM tetracaine were run after every ninth assay plate. The response toNa⁺-dependent depolarization was measured and the data were binned intohistograms as shown. The mean maximum response (Max) obtained in thepresence of (0.1% DMSO) and the mean minimum response (Min) obtained inthe presence of 10 μM tetracaine were determined. For quality control,data variance was calculated using a Z′ factor method that compares thedifference between the maximum and minimum signals in order todiscriminate hit compounds from the background variation, see, e.g.,Ji-Hu Zhang et al, A Simple Statistical Parameter for Use in Evaluationand Validation of High Throughput Screening Assays, 4(2) J. Biomol.Screen. 67-73 (1999). This was accomplished by calculating a screeningwindow (z) for each control plate. Data for the run was accepted if1.0≧Z≧0.5. The Z′ factor is calculated by comparing the difference ofthe means of a positive and negative control with their respectivestandard deviations as in equation:

$Z = {1 - \frac{{3 \times {STD}_{\max}} + {3 \times {STD}_{\min}}}{{Mean}_{\max} - {Mean}_{\min}}}$

Example 2 Screening Assay for Identifying Persistent Sodium CurrentBlockers using Single Wavelength Voltage-Sensitive Dyes

To establish an assay plate HEK-293 cells stably transfected with a cDNAfor the Na_(v)1.3 sodium channel capable of mediating a persistentsodium current (HEK-Na_(v)1.3 cells) were grown in Minimum EssentialMedium (Invitrogen, Inc., Carlsbad, Calif.) supplemented with 10% FetalBovine Serum (Invitrogen, Inc., Carlsbad, Calif.), 1%Penicillin-Streptomycin (Invitrogen, Inc., Carlsbad, Calif.), 500 mg/mLG418 Geneticin (Invitrogen, Inc., Carlsbad, Calif.) and 2 μM TTX(Calbiochem, Inc., San Diego, Calif.) for maintaining selectivepressure. Cells were grown in vented cap flasks, in 90% humidity and 10%CO₂, to about 80% confluence, harvested by trypsinization and celldensity was determined. Approximately 16 to 24 hours before the assay,cells were seeded at 75,000 cells per well in 150 μL of MEM media inclear bottom, black-wall, poly-d-lysine coated 96-well plates (BDBiosciences) and stored in a 5% CO₂, 37° C. incubator overnight. Thisplating procedure resulted in an optimal cell confluence (80%-90%) atthe time of the assay.

To examine the ability of test molecules to alter persistent sodiumcurrent, the medium was aspirated from the wells and replaced with 40 mLof TEA-MeSO₃ (Na⁺ depletion) solution containing the following:TEA-MeSO₃ (140 mM), HEPES-MeSO₃ (10 mM), KMeSO₃ (4.5 mM), Glucose (10mM) MgCl₂ (1 mM), CaCl₂ (1 mM), CdCl₂ (0.2 mM) with a pH of 7.4 and anosmolarity of 300-310 mOsm. The Na⁺ depletion solution also contained 4×of the final test concentration of Molecular Devices membrane potentialdye (Molecular Devices Corp., Sunnyvale, Calif.) made up according tomanufacturers instructions. The Molecular Devices membrane potential dyeis a lipophilic, anionic, bis-oxonol dye that can partition across thecytoplasmic membrane of live cells, dependent on the membrane potentialacross the plasma membrane. Its fluorescence intensity increases whenthe dye is bound to cytosolic proteins. When the cells are depolarized,more dye enters the cells, and the increased intracellular concentrationof the dye binding to intracellular lipids and proteins causes anincrease in fluorescence signal. When the cells are hyperpolarized, dyeexits the cells, and the decreased intracellular concentration of dyebinding to lipids and proteins results in a decreased of fluorescencesignal. The dye was excited at the 488 nm wavelength. At this timeeither positive or negative control compounds or test molecules wereadded to the wells of the plate at 1× their final test concentration.The plate was allowed to incubate with the dye and compounds wereallowed to incubate for about 25-30 minutes at room temperature in dark.

The assay plates were then transferred to a Fluorometric Imaging PlateReader (FLIPR-Tetra, Molecular Devices Corp., Sunnyvale, Calif.) formeasurement of depolarization induced by addition of the Na⁺ repletionbuffer. The Na⁺ repletion buffer solution comprised the following:NaMeSO₃ (140 mM), HEPES-MeSO₃ (10 mM), KMeSO₃ (13 mM), Glucose (10 mM)MgCl₂ (1 mM), CaCl₂ (1 mM), CdCl₂ (0.2 mM) with a pH of 7.4 and anosmolarity of 300-310 mOsm. The Na⁺ depletion solution also contained 1×of the final test concentration of either positive or negative controlcompounds or test molecules.

The parameters for the FLIPR-Tetra data acquisition were set as follows:the excitation wavelength was set to a bandpass of 510-545 nm; theemission wavelength was set to a bandpass of 565-625 nm; the gain ofcamera was set between 60-100 with an exposure time of 0.1 s and with anacquisition rate of 5 Hz.

The assay protocol was as follows: after transferring the plates to theFLIPR-Tetra baseline fluorescence was measured for 5 sec at which time120 μL of the Na⁺ repletion buffer was added to initiate a depolarizingresponse (FIG. 14A, Control). This response contained both a specificdepolarization a result of Na⁺ flux across voltage-gated sodium channels(VGSC) and a non-specific depolarization resulting from otherNa⁺-dependent mechanisms in the HEK cells. The background response wasrevealed in wells that contained an excess of TTX (1 μM) to block allVGSC. Specific responses were measured by subtracting the averageresponse of wells containing 1 μM TTX from the test wells (FIG. 14B).

For data analysis the peak fluorescence amplitude measures from eachwell of the test plates in the FLIPR were calculated automatically byScreenworks (Molecular Devices Corp, Sunnyvale, Calif.) which determinedthe maximum peak as the difference from the most positive peak relativeto baseline. Peak amplitude measures were then imported to an exceltemplate file which is used to calculate mean and SD. Mean amplitudemeasures from each drug-treated group were normalized with respect tothe mean of control group. Normalized mean amplitude measures fromcontrol and drug treated wells were imported into Origin for plottingconcentration-response curves of and determination IC₅₀ values ofpersistent sodium channel blockers.

To test the accuracy and reproducibility of the Na⁺ depletion/repletionassay on the FLIPR system, two assay formats were used: a screeningwindow format to measure the ability to obtain reproducible data in thesingle-concentration or HTS mode of screening (FIG. 15A) and adose-response format to measure the ability to accurately predict IC₅₀of known reference compounds (FIG. 15B)

Results from 96-well plate setup in the screening window formatdemonstrated the overall reproducibility of the response (FIG. 16). Inthis plate Na⁺ was added to wells A1-H6 to induce depolarization while 1μM TTX were incubated for 30 minutes in wells A7-H12 prior to Na⁺addition. The Na⁺ channel dependent component of the fluorescenceresponse in wells A7-H12 was blocked by TTX.

Within HTS assays a standard method to evaluate the ability todiscriminate hit compounds from the background variation in thesignal-to-noise ratio of the assay was calculated using a metric calledZ′ factor, see, e.g., Ji-Hu Zhang et al, A Simple Statistical Parameterfor Use in Evaluation and Validation of High Throughput ScreeningAssays, 4(2) J. Biomol. Screen. 67-73 (1999). The Z′ factor wascalculated by comparing the difference of the means of a positive andnegative control with their respective standard deviations as inequation:

$Z = {1 - \frac{{3 \times {STD}_{\max}} + {3 \times {STD}_{\min}}}{{Mean}_{\max} - {Mean}_{\min}}}$

Mean Fluorescence peak amplitude and standard deviation from control anddrug treated wells was used to calculate the screening window factor(Z′). Peak fluorescence amplitude measures from each well was calculatedautomatically by Screenworks which determined the maximum peak as thedifference from the most positive peak relative to baseline. Peakamplitude measures were then imported to an excel template file whichwas used to calculate mean and SD. Mean and SD from control and drugtreated groups were input to equation 1 for determination of screeningwindow factor (Z′). For the data illustrated in FIG. 15 the Z′ factorwas 0.52 (FIG. 16B). Assays were generally considered acceptable when z′varies between 0.5 and 1.0.

To examine the relative potency of test molecules against persistentsodium currents their IC₅₀ was determined from dose response data asshown in FIG. 17. In this the assay plate in FIG. 17A, wells A1-H2, andA11-H12 received Na⁺ addition while other wells from column 3-10 weretreated with TTX ranging from 0.5 nM to 1000 nM (see plate layout inFIG. 15B). The average fluorescence waveforms from control and TTXtreated groups were plotted to illustrate the dose dependent blockade ofNa⁺-induced fluorescence response by TTX (FIG. 17B). Peak amplitudemeasures from the reduced data were exported to an Excel analysistemplate file which calculated mean fluorescence amplitude and SD. Meanamplitude measures from test-molecule treated groups were normalizedwith respect to the mean of control group. Normalized mean amplitudemeasures from control and TTX treated wells were imported into Originfor determination of IC₅₀. Normalized mean amplitudes were plotted as afunction of dose response of TTX in log scale. IC₅₀ was determined bycurve fitting using the logistic dose response equation:

$Y = {\frac{A_{1} - A_{2}}{1 + \left( {x/x_{0}} \right)^{p}} + A_{2}}$

where

-   -   x₀=center    -   p=power    -   A₁=Initial Y value    -   A₂=Final Y value    -   The Y value at x₀ is half way between the two limiting values A₁        and A₂

Y(x₀)=(A₁+A₂)/2

Concentration-response curves obtained from three test compounds areshown in FIG. 17C. The IC₅₀ values for TTX, Tetracaine and Lidocaine of5 nM, 1.8 μM and 32 μM obtained in this assay correspond well with thevalues obtained using patch-clamp (5 nM, 1 μM and 90 μM respectively).

Example 3 Screening Assay for Identifying Transient Sodium CurrentBlockers using Electric Field Stimulation (EFS)

Methods for applying external electric fields to stimulate excitablecells and tissues are well known and have been extensively reviewed,see, e.g., Peter J. Basser and Bradley J. Roth, New Currents inElectrical Stimulation of Excitable Tissues, 2 Annu. Rev. Biomed. Eng.377-397 (2000); Jaakko Malmivuo and Robert Plonsey, Bioelectromagnetism:Principles and Applications of Bioelectric and Biomagnetic Fields,(Oxford University Press, New York. 472 pp. 1995); and J. PatrickReilly, Electrical Stimulation and Electropathology, (CambridgeUniversity Press, Cambridge.1-522 pp. 1992).

To establish an assay to measure the potency of compounds for blockingtransient sodium currents in order to compare their potency againstblocking persistent sodium currents, HEK-293-Na_(v)1.3 cells will begrown in Minimum the presence of 500 mg/mL G418 Geneticin (Invitrogen,Inc., Carlsbad, Calif.) and 2 μM TTX (Calbiochem, Inc., San Diego,Calif.) to maintain selective pressure.

To measure transient currents cells will be transferred to a recordingchamber suitably instrumented with electrodes to produce EFS asdescribed in, e.g., Michael P. Maher & Jesus E. Gonzalez, Multi-wellPlate and Electrode Assemblies for Ion Channel Assays, U.S. Pat. No.6,969,449 (Nov. 29, 2005); Michael P. Maher & Jesus E. Gonzalez, HighThroughput Method and System for Screening Candidate Compounds forActivity Against Target Ion Channels, U.S. Pat. No. 6,686,193 (Feb. 3,2004). and Paul Burnett et al., Electrophysiology Assay Methods, U.S.Patent Publication No. 2004/0115614 (Jun. 17, 2004).

Cells will be loaded with either an appropriate FRET donor/acceptorvoltage-sensitive dye pair as described in Example 1 or a singlewavelength voltage-sensitive dye as described in Example 2. The cellswill be transferred to appropriate device to record membrane potentialinduced changes in fluorescence, e.g., a VIPR (Aurora Bioscience, SanDiego, Calif.) or FLIPR-tetra (Molecular Devices, Sunnyvale, Calif.).

Optical measurement of fluorescent changes in response of EFS will bemeasured of a series of stimuli. The transient Na+ current producesrapid change in fluorescence due to the rapid depolarization. Forquantification of the block of transient current, the amplitude of peakresponse will be averaged form a series of stimuli. The average responsewill be converted to activity by normalizing against the differencebetween the responses in Ringer's solution with DMSO and Ringer'ssolution containing 10 μM tetracaine or 100 nM TTX. Normalized activityagainst the transient current will be plotted as a concentration doseresponse curve and IC₅₀ for block against transient currents can becalculated by a fitting a logistic function to the data.

Example 4 Screening Assay for Identifying Transient Sodium CurrentBlockers Using Automated Patch-Clamp Technology

HEK-293 cells stably transfected with a cDNA for the Na_(v)1.3 sodiumchannel capable of mediating a persistent sodium current (HEK-Na_(v)1.3cells) were grown in Minimum Essential Medium (Invitrogen, Inc.,Carlsbad, Calif.) supplemented with 10% Fetal Bovine Serum (Invitrogen,Inc., Carlsbad, Calif.), 1% Penicillin-Streptomycin (Invitrogen, Inc.,Carlsbad, Calif.), 500 mg/mL G418 Geneticin (Invitrogen, Inc., Carlsbad,Calif.) and 2 μM TTX (Calbiochem, Inc., San Diego, Calif.) formaintaining selective pressure. Cells were grown in vented cap flasks,in 90% humidity and 10% CO₂, to about 80% confluence, harvested bytrypsinization and cell density was determined. Cells were resuspendedat a density of 2×10⁶/mL in the extracelluar solution described belowand transferred to either a IonWorks (Molecular Devices, Sunnyvale,Calif.) or Flyscreen (Flyion, GmbH) automated patch clamp formeasurement of peak transient Na⁺ current.

Solutions used for these experiments were as follows: Internal Solution(in mM): 140 KCl, 2 MgCl₂ 5 EGTA, 10 Hepes pH to 7.2 with KOH; ExternalSolution (in mM): 137 NaCl, 4 KCl, 1 MgCl₂, 1.8 CaCl₂, 10 Hepes, 10Glucose, pH to 7.4 with NaOH.

Na⁺ currents were elicited by the following pulse protocol: Cells wereheld at a −90 mV and stimulated every 5 sec with a voltage step to 0 mVfor 25 ms. Peak inward current was measured between 1 and 5 ms after theonset of the voltage pulse. After a pre-addition run to determinebaseline currents, compounds were applied by and allowed to incubate for5 minutes and a second reading was then taken to compare currents in thepresent of either positive or negative control compounds or testcompounds. FIG. 18 shows an example of four such experiments from anIonWorks automated patch clamp. The peak currents from the pre-additionrun and after exposure to 100 nM TTX are labeled.

To examine the relative potency of test molecules against transientsodium currents their IC₅₀ was determined from dose response data asshown in FIG. 19. Peak current measures from the reduced data shown inFIG. 18 were exported to an Excel analysis template file whichcalculated mean current amplitude and SD. Mean amplitude measures fromtest-molecule treated groups were normalized with respect to the mean ofcontrol group. Normalized mean amplitude measures from control and TTXtreated wells were imported into Origin for determination of IC₅₀.Normalized mean amplitudes were plotted as a function of dose responseof TTX in log scale. IC₅₀ was determined by curve fitting using thelogistic dose response equation:

$Y = {\frac{A_{1} - A_{2}}{1 + \left( {x/x_{0}} \right)^{p}} + A_{2}}$

where

-   -   x₀=center    -   p=power    -   A₁=Initial Y value    -   A₂=Final Y value    -   The Y value at x₀ is half way between the two limiting values A₁        and A₂    -   Y(x₀)=(A₁+A₂)/2

IC₅₀ values obtained in this assay were then be compared against IC₅₀for blocking persistent currents as measured in examples 1 and 2. Thisallows the calculation of the relative selectivity of block forpersistent vs. transient currents.

Example 5 Electrophysiological Assay for Selectivity of Inhibitors ofPersistent Sodium Current

To confirm the blocking selectivity of test compounds for persistentsodium current, individual compounds were examined using a whole-cellpatch clamp method. HEK cells transfected with Na_(v)1.3 sodium channelsthat express transient and persistent sodium currents were plated ontoglass coverslips and cultured in MEM cell culture media with Earle'ssalts and GlutaMAX (Invitrogen, Inc., Carlsbad, Calif.) supplementedwith: 10% Fetal bovine serum, heat inactivated (Invitrogen, Inc.,Carlsbad, Calif.), 0.1 mM MEM non-essential amino acids (Invitrogen,Inc., Carlsbad, Calif.), 10 mM HEPES (Invitrogen, Inc., Carlsbad,Calif.), 1% Penicillin/Streptomycin (Invitrogen, Inc., Carlsbad,Calif.).

After an incubation period of from 24 to 48 hours the culture medium wasremoved and replaced with external recording solution (see below). Wholecell patch clamp experiments were performed using an EPC10 amplifier(HEKA Instruments, Lambrecht, Germany.) linked to an IBM compatiblepersonal computer equipped with PULSE software. Borosilicate glass patchpipettes were pulled to a fine tip on a P90 pipette puller (SutterInstrument Co., Novato, Calif.) and were polished (Microforge,Narishige, Japan) to a resistance of about 1.5 Mohm when filled withintracellular recording solution (Table 3).

Persistent and transient currents in HEK cells expressing Na_(v)1.3channels were measured by applying 200-msec depolarizations from aholding potential of −90 mV to 0 mV. Background currents that remainedin the presence of 500 nM TTX were subtracted from all traces. Drugswere perfused directly into the vicinity of the cells using amicroperfusion system.

TABLE 3 Patch Clamp Solutions External Recording Solution InternalRecording Solution Compound Concentration Compound Concentration NaCl127 mM CsMeSO₃ 125 mM HEPES (free acid) 10 mM CsCl 25 mM KCl 5 mMNaHEPES 10 mM CsCl 5 mM Amphotericin 240 μg/mL Glucose 10 mM MgCl₂ 0.6mM CaCl₂ 1.2 mM CdCl₂ 200 μM pH to 7.4 with NaOH @ room temp. pH 7.20with CsOH300 mOsm 290 mOsm.

Under control conditions, depolarizing pulses elicited a large transientinward current that declined to a smaller persistent current, whichremained stable during the remainder of the pulse (FIG. 12, control).Addition of 500 nM TTX completely blocked both the transient andpersistent currents (FIG. 12, TTX). Application of 3 μM of Compound 1produced a much different effect. Inspection of FIG. 12 reveals that theCompound 1 blocked 99% of the persistent current while only reducing thetransient current by 16%. Dose-response analysis for Compound 1demonstrates its significant selectivity for blocking the persistentsodium current relative to the transient sodium current over a fourorder of magnitude range (FIG. 13).

1. A method for identifying a selective blocker of a persistent Na⁺channel whereby the method comprises the steps of: a) providing a testsample 1 comprising i) a Cl⁻-free physiological solution; ii) avoltage-sensitive fluorescence dye; iii) a cell having a K⁺ channel anda persistent Na⁺ channel wherein a K⁺ conductance of the K⁺ channel isat least 50-fold higher than a Na⁺ conductance from the persistent Na⁺channel; and iv) a potential Na⁺ channel blocker; b) depolarizingmembrane of the cell with a Na/K pump blocker to the test sample 1, theNa/K pump blocker selected from the group consisting of an oubainderivative, an isothiouronium or derivative thereof, a digitoxigenin orderivative thereof, a coumestan or derivative thereof, a vanadate orderivative thereof, a cardenolide or derivative thereof and a naturalcardiac glycoside or derivative thereof; c) detecting fluorescenceemitted by the voltage-sensitive dye in test sample 1; d) providing acontrol sample 1 comprising i) a Cl⁻-free physiological solution; ii) avoltage-sensitive fluorescence dye; and iii) a cell having a K⁺ channeland a persistent Na⁺ channel wherein a K⁺ conductance of the K⁺ channelis at least 50-fold higher than a Na⁺ conductance from the persistentNa⁺ channel; e) depolarizing membrane of the cell with a Na/K pumpblocker to the control sample 1; f) detecting fluorescence emitted bythe voltage-sensitive dye in the control sample 1; g) comparing theemitted fluorescence from step (c) to the emitted fluorescence from step(f); h) providing a test sample 2 comprising i) a physiologicalsolution; ii) a voltage-sensitive fluorescence dye; iii) a cell having aK⁺ channel and a transient Na⁺ channel; and iv) a potential Na⁺ channelblocker i) depolarizing membrane of the cell in test sample 2; j)detecting the fluorescence emitted by the voltage-sensitive dye in testsample 2; k) providing a control sample 2 comprising i) a physiologicalsolution; ii) a voltage-sensitive fluorescence dye; and iii) a cellhaving a K⁺ channel and a transient Na⁺ channel; l) depolarizingmembrane of the cell in control sample 2; m) detecting the fluorescenceemitted by the voltage-sensitive dye in control sample 2; n) comparingthe emitted fluorescence from step (j) relative to an emittedfluorescence from step (m); o) comparing the difference in step (g) withthe difference in step (n).
 2. The method according to claim 1, whereinthe Na/K pump blocker is an oubain derivative.
 3. The method accordingto claim 1, wherein the Na/K pump blocker is an isothiouronium orderivative thereof.
 4. The method according to claim 1, wherein the Na/Kpump blocker is a digitoxigenin or derivative thereof.
 5. The methodaccording to claim 1, wherein the Na/K pump blocker is a coumestan orderivative thereof.
 6. The method according to claim 1, wherein the Na/Kpump blocker is a vanadate or derivative thereof.
 7. The methodaccording to claim 1, wherein the Na/K pump blocker is a cardenolide orderivative thereof.
 8. The method according to claim 1, wherein the Na/Kpump blocker is a natural cardiac glycoside or derivative thereof. 9.The method according to claim 1, wherein the cell expresses a persistentNa⁺ channel selected from the group consisting of Na_(v)1.3, Na_(v)1.5,Na_(v)1.6 and Na_(v)1.9.
 10. A method for identifying a blocker of apersistent Na⁺ channel whereby the method comprises the steps of: a)providing a test sample 1 comprising i) a Cl⁻-free physiologicalsolution; ii) a voltage-sensitive fluorescence dye; iii) a cell having aK⁺ channel and a persistent Na⁺ channel wherein a K⁺ conductance of theK⁺ channel is at least 50-fold higher than a Na⁺ conductance from thepersistent Na⁺ channel; and iv) a potential Na⁺ channel blocker; b)depolarizing membrane of the cell with a Na/K pump blocker to the testsample 1, the Na/K pump blocker selected from the group consisting of anoubain derivative, an isothiouronium or derivative thereof, adigitoxigenin or derivative thereof, a coumestan or derivative thereof,a vanadate or derivative thereof, a cardenolide or derivative thereofand a natural cardiac glycoside or derivative thereof; c) detectingfluorescence emitted by the voltage-sensitive dye in test sample 1; d)providing a control sample 1 comprising i) a Cl⁻-free physiologicalsolution; ii) a voltage-sensitive fluorescence dye; and iii) a cellhaving a K⁺ channel and a persistent Na⁺ channel wherein a K⁺conductance of the K⁺ channel is at least 50-fold higher than a Na⁺conductance from the persistent Na⁺ channel; e) depolarizing membrane ofthe cell with a Na/K pump blocker to the control sample 1; f) detectingfluorescence emitted by the voltage-sensitive dye in the control sample1; g) comparing the emitted fluorescence from step (c) relative to theemitted fluorescence from step (f).
 11. The method according to claim10, wherein the Na/K pump blocker is an oubain derivative.
 12. Themethod according to claim 10, wherein the Na/K pump blocker is anisothiouronium or derivative thereof.
 13. The method according to claim10, wherein the Na/K pump blocker is a digitoxigenin or derivativethereof.
 14. The method according to claim 10, wherein the Na/K pumpblocker is a coumestan or derivative thereof.
 15. The method accordingto claim 10, wherein the Na/K pump blocker is a vanadate or derivativethereof.
 16. The method according to claim 10, wherein the Na/K pumpblocker is a cardenolide or derivative thereof.
 17. The method accordingto claim 10, wherein the Na/K pump blocker is a natural cardiacglycoside or derivative thereof.
 18. The method according to claim 10,wherein the cell expresses a persistent Na⁺ channel selected from thegroup consisting of Na_(v)1.3, Na_(v)1.5, Na_(v)1.6 and Na_(v)1.9.